Oligonucleotides containing 2'-deoxy-2'-F-2'-C-methyl nucleotides

Abstract

The present disclosure relates generally to 2'-geminally substituted nucleosides and 2'-geminally substituted nucleotides, and oligonucleotides and dsRNA molecules that comprise such 2'-geminally substituted nucleosides and 2'-geminally substituted nucleotides. TIFF2024539093000234.tif77159

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under Section 119(e) of U.S. Provisional Application No. 63 / 257,289, filed October 19, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Technical Field FIELD OF THE DISCLOSURE The present disclosure relates generally to 2'-geminal substituted nucleosides, oligonucleotides and dsRNAs comprising same, and uses thereof. [Background technology]

[0003] background RNA interference, or "RNAi," was first coined by Fire and colleagues to describe the observation that double-stranded RNAi (dsRNA) can block gene expression (Fire et al. (1998) Nature 391, 806-811; Elbashir et al. (2001) Genes Dev. 15, 188-200). Short dsRNAs direct gene-specific posttranscriptional silencing in many organisms, including vertebrates, and have become a new tool for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from double-stranded RNA triggers, but the protein components of this activity remained unknown.

[0004] There remains a need in the art for effective nucleotide or chemical modifications for dsRNA molecules that are advantageous for inhibiting target gene expression, and the present invention is directed to that endeavor. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Fire et al. (1998) Nature 391, 806-811, Elbashir et al. (2001) Genes Dev. 15, 188-200 Summary of the Invention

[0006] overview In one aspect, provided herein are (i) at least one 2'-geminal substituted nucleoside of formula (I) or (I'): TIFF2024539093000002.tif33128, and / or (ii) a 2'-geminal substituted nucleoside of formula (II) or (II') at the 5'-terminal nucleotide: Oligonucleotides are provided, including TIFF2024539093000003.tif33128.

[0007] In formulas (I), (I'), (II) and (II'), X is O, S, C(R X )2, or N(R XN ) and Each R X are independently hydrogen, halogen, or optionally substituted C 1~4 Alkyl, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, or optionally substituted C 2~4 alkynyl, or both R X Overall, =O, =S, =N(R N ), or =CH2, R XN is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 1~30alkyl-COH, or a nitrogen protecting group, B is an optionally modified nucleobase; R a' is hydrogen, halogen, -OR a2 , -SR a3 , optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 Alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH2CH2O) m CH2CH2OR a4 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH2CH2NH) n CH2CH2-R a5 , NHC(O)R a4 , a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, a solid support, a linker covalently attached to a solid support, or an internucleoside linkage to a subsequent nucleotide; R a2 is a hydrogen or hydroxyl protecting group, R a3 is a hydrogen or sulfur protecting group, R a4 independently for each occurrence, H, C 1~ C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R a5 and R a5 is independently at each occurrence amino (NH), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino; m is 1 to 50; n is 1 to 50; R b may be substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 alkynyl, or halogen; R c is the bond to the internucleoside linkage to the subsequent nucleotide, hydrogen, halogen, -OR c2 , -SR c3 , optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 Alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH2CH2O) r CH2CH2OR c4 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH2CH2NH) s CH2CH2-R c5 , NHC(O)R c4 , a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, a solid support, or a linker covalently attached to a solid support, optionally comprising at least R c or R b is the bond to the internucleoside linkage to the subsequent nucleotide, R c2 is a hydrogen or hydroxyl protecting group, R c3 is a hydrogen or sulfur protecting group, R c4 are independently generated for each occurrence, H, C1 to C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R c5 and R c5is independently at each occurrence amino (NH), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino; r is 1 to 50; s is 1 to 50; R 4 is hydrogen, optionally substituted C 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, or optionally substituted C 1~6 Is it an alkoxy? Alternatively, R 4 and R a is, as a whole, 4'-C(R a11 R a12 ) v -Y-2' or 4'-YC(R a11 R a12 ) v -2', Y is -O-, -CH2-, -CH(Me)-, -C(CH3)2-, -S-, -N(R a13 )-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -OC(O)-, -C(O)O-, -N(R a13 )C(O)-, or -C(O)N(R a13 )- and R a11 and R a12 are independently H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl; R a13 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 1~30 alkyl-COH, or a nitrogen protecting group, v is 1, 2 or 3, Alternatively, R 4 and R c C, together with the atoms to which they are attached, may be substituted 3~8 Cycloalkyl, optionally substituted C 3~8 forming a cycloalkenyl or an optionally substituted 3- to 8-membered heterocyclyl, R d is -CH(R d1 )-R d2 or -C(R d1 )=CHR d2 and R d1 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted -C 2~30 alkenyl, or optionally substituted -C 2~30 is alkynyl, R d2 is the bond to the internucleoside linkage to the preceding nucleotide, R e may be substituted C 1~6 Alkyl-R e1 , optionally substituted -C 2~6 Alkenyl-R e1 , or optionally substituted -C 2~6 Alkynyl-R e1 and R e1 -OR e2 , -SR e3 , -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -P(S)(SR e5 )2, -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(O)(OR e4 )2, -SP(S)(OR e4 )2, -SP(S)(SR e5 )(ORe4 ), or -SP(S)(SR e5 )2, R e2 is a hydrogen or oxygen protecting group, R e3 is a hydrogen or sulfur protecting group, Each R e4 are independently hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, or optionally substituted C 2~30 alkynyl, or oxygen protecting group; And each R e5 are independently hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, or optionally substituted C 2~30 alkynyl, or sulfur protecting groups.

[0008] In another aspect, provided herein is a double-stranded nucleic acid (e.g., dsRNA) comprising a first strand and a second strand substantially complementary to the first strand, wherein at least one of the first strand or the second strand is an oligonucleotide described herein, e.g., an oligonucleotide comprising (i) at least one 2'-geminal substituted nucleoside of Formula (I) or (I'), and / or (ii) a 2'-geminal substituted nucleoside of Formula (II) or (II') at the 5'-terminal nucleotide. In some embodiments, the first strand is an oligonucleotide described herein. In some embodiments of any one of the aspects described herein, the oligonucleotide comprises (i) at least one 2'-geminal substituted nucleoside of Formula (I) and / or (ii) a 2'-geminal substituted nucleoside of Formula (II) at the 5'-terminal nucleotide.

[0009] In some embodiments of any one of the aspects described herein, the double-stranded nucleic acid (e.g., dsRNA) comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand are complementary to each other to form a double-stranded region, e.g., a double-stranded region of at least 15 base pairs. The antisense strand comprises (a) a 5'-terminal nucleoside that is a 2'-geminal substituted nucleoside of Formula (II) or (II'), and (b) at least one or both of 2'-geminal substituted nucleosides of Formula (I) or (I') at one of positions 2 to 9 (e.g., positions 2, 3, 4, 5, 6, 7, 8, and / or 9) counting from the 5' end of the antisense strand.

[0010] In some embodiments of any one of the aspects described herein, the antisense strand includes (i) a 5'-terminal nucleoside that is a 2'-geminal substituted nucleoside of Formula (II), and / or (ii) at least a 2'-geminal substituted nucleoside of Formula (I) at one of positions 2-9 (e.g., positions 2, 3, 4, 5, 6, 7, 8, and / or 9), counting from the 5' end of the antisense strand.

[0011] In some embodiments of any one of the aspects described herein, the 5'-terminal nucleotide of the antisense strand is a 2'-geminal substituted nucleotide of Formula (II) or (II'). For example, the 5'-terminal nucleotide of the antisense strand is a 2'-geminal substituted nucleotide of Formula (II).

[0012] In some embodiments of any one of the aspects described herein, the antisense strand comprises a vinyl phosphonate (e.g., E-vinyl phosphonate) group at its 5'-end. For example, the 5'-terminal nucleotide of the antisense strand is a 2'-geminally substituted nucleotide of formula (II) or (II'), where R e is a vinyl phosphonate (e.g., R e is R e is -CH=CHR e1 and R e1 -P(O)(OR e4In some embodiments of any one of the aspects described herein, the 5'-terminal nucleotide of the antisense strand is a 2'-geminally substituted nucleotide of Formula (II), wherein R e is a vinyl phosphonate (e.g., R e is R e is -CH=CHR e1 and R e1 -P(O)(OR e4 )2).

[0013] In some embodiments of any one of the aspects described herein, the antisense strand comprises a nucleoside of Formula (I) or (I') at least at position 3, 4, 5, 6, 7, 8, or 9, counting from the 5' end of the antisense strand. For example, the antisense strand comprises a nucleoside of Formula (I) or (I') at least at position 4, counting from the 5' end of the antisense strand. In another example, the antisense strand comprises a nucleoside of Formula (I) or (I') at least at position 5, counting from the 5' end of the antisense strand. In yet another example, the antisense strand comprises a nucleoside of Formula (I) or (I') at least at position 6, counting from the 5' end of the antisense strand. In yet another example, the antisense strand comprises a nucleoside of Formula (I) or (I') at least at position 7, counting from the 5' end of the antisense strand. In yet another example, the antisense strand comprises a nucleoside of Formula (I) or (I') at least at position 8, counting from the 5' end of the antisense strand. In one example, the antisense strand comprises a nucleoside of Formula (I) or (I') at least at position 9, counting from the 5' end of the antisense strand.

[0014] In some embodiments of any one of the aspects described herein, the antisense strand comprises a nucleoside of Formula (I) at least at position 3, 4, 5, 6, 7, 8, or 9, counting from the 5' end of the antisense strand. For example, the antisense strand comprises a nucleoside of Formula (I) at least at position 4, counting from the 5' end of the antisense strand. In another example, the antisense strand comprises a nucleoside of Formula (I) at least at position 5, counting from the 5' end of the antisense strand. In yet another example, the antisense strand comprises a nucleoside of Formula (I) at least at position 6, counting from the 5' end of the antisense strand. In yet another example, the antisense strand comprises a nucleoside of Formula (I) at least at position 7, counting from the 5' end of the antisense strand. In yet another example, the antisense strand comprises a nucleoside of Formula (I) at least at position 8, counting from the 5' end of the antisense strand. In one example, the antisense strand comprises a nucleoside of Formula (I) at least at position 9, counting from the 5' end of the antisense strand.

[0015] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (I) has Formula (IA): The file is TIFF2024539093000004.tif28128.

[0016] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (I) has the formula (IB): TIFF2024539093000005.tif28128.

[0017] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II) has Formula (IIA): TIFF2024539093000006.tif28128.

[0018] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II) has Formula (IIB): The file is TIFF2024539093000007.tif28128.

[0019] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (I') has Formula (IA'): TIFF2024539093000008.tif25128.

[0020] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (I') has Formula (IB'): TIFF2024539093000009.tif25128.

[0021] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II') has Formula (IIA'): The file is TIFF2024539093000010.tif28128.

[0022] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II') has Formula (IIB'): The file is TIFF2024539093000011.tif28128.

[0023] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II) is a nucleoside of Formula (IIA) or (IIB): During the ceremony, X is O, R a' is a halogen (e.g., F or Cl), hydroxyl, optionally substituted C 1~30 Alkoxy (e.g., -(CH-22) n CH3, where n is 1 to 21, for example, 1, 16, or -(CH22) m -NH2, where m is 2 to 10, for example 3 or 6, or -(CH22) p -OMe, where p is 1 to 21, e.g., 1 or 2), or a bond to an internucleoside linkage to a subsequent nucleoside; R b may be substituted C 1~6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl); R c is the bond or hydroxyl to the internucleoside linkage to the subsequent nucleoside, provided that R a' and R c only one of which is the bond to the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R e is -CH=CHR e1 where R e1 is -P(O)(OR e4 )2.

[0024] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II) is a nucleoside of Formula (IIA) or (IIB): During the ceremony, X is O, R a' is F, Cl or optionally substituted C 1~30 Alkoxy (e.g., -(CH22) n CH3, where n is 1 to 21, for example, 1, 16, or -(CH22) m -NH2, where m is 2 to 10, for example 3 or 6, or -(CH22) p -OMe, where p is 1 to 21, for example 1 or 2; R b may be substituted C 1~6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl); R c is the bond to the internucleoside linkage to the subsequent nucleoside, R4 is hydrogen, and R e is -CH=CHR e1 where R e1 is -P(O)(OR e4 )2.

[0025] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II) is a nucleoside of Formula (IIA) or (IIB): During the ceremony, X is O, R a' is Cl or optionally substituted C 1~30 Alkoxy (e.g., -(CH22) n CH3, where n is 1 to 21, for example, 1, 16, or -(CH22) m -NH2, where m is 2 to 10, for example 3 or 6, or -(CH22) p -OMe, where p is 1 to 21, for example 1 or 2; R b may be substituted C 1~6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl); R c is the bond to the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R e is -CH2-OR e2 or -CH=CHR e1 where R e2 is a hydrogen or oxygen protecting group, and R e1 is -P(O)(OR e4 )2.

[0026] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II) is a nucleoside of Formula (IIA) or (IIB): During the ceremony, X is O, R a' is a halogen (e.g., F, Br, or Cl), R b is R a' is a halogen (e.g., F, Br, or Cl), R c is the bond or hydroxyl to the internucleoside linkage to the subsequent nucleoside, provided that R a' and R c only one of which is the bond to the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R e is -CH2-OR e2 or -CH=CHR e1 where R e2 is a hydrogen or oxygen protecting group, and R e1 is -P(O)(OR e4 )2.

[0027] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II') is a nucleoside of Formula (IIA') or (IIB'): During the ceremony, X is O, R a' is a halogen (e.g., F, Br, or Cl), hydroxyl, optionally substituted C 1~30 Alkoxy (e.g., -(CH22) n CH3, where n is 1 to 21, for example, 1, 16, or -(CH22) m -NH2, where m is 2 to 10, for example 3 or 6, or -(CH22) p -OMe, where p is 1 to 21, e.g., 1 or 2), or a bond to an internucleoside linkage to a subsequent nucleoside; R b may be substituted C 1~6alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl) or halogen (e.g., F, Br, or Cl); R c is the bond or hydroxyl to the internucleoside linkage to the subsequent nucleoside, provided that R a' and R c only one of which is the bond to the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R e is -CH2-OR e2 or -CH=CHR e1 where R e2 is a hydrogen or oxygen protecting group, and R e1 -P(O)(OR e4 )2.

[0028] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (I) is a nucleoside of Formula (IA) or (IB): During the ceremony, X is O, R a' is a halogen (e.g., F or Cl), hydroxyl, optionally substituted C 1~30 Alkoxy (e.g., -(CH-22) n CH3, where n is 1 to 21, for example, 1, 16, or -(CH22) m -NH2, where m is 2 to 10, for example 3 or 6, or -(CH22) p -OMe, where p is 1 to 21, e.g., 1 or 2), or a bond to an internucleoside linkage to a subsequent nucleoside; R b may be substituted C 1~6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl); R c is the bond or hydroxyl to the internucleoside linkage to the subsequent nucleoside, provided that R a' and R c only one of which is the bond to the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R d is the bond to the internucleoside linkage to the preceding nucleoside.

[0029] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (I) is a nucleoside of Formula (IA) or (IB), During the ceremony, X is O, R a' is F, Cl or optionally substituted C 1~30 Alkoxy (e.g., -(CH22) n CH3, where n is 1 to 21, for example, 1, 16, or -(CH22) m -NH2, where m is 2 to 10, for example 3 or 6, or -(CH22) p -OMe, where p is 1 to 21, for example 1 or 2; R b may be substituted C 1~6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl); R c is the bond or hydroxyl at the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R d is the bond to the internucleoside linkage to the preceding nucleoside.

[0030] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (I) is a nucleoside of Formula (IA) or (IB), During the ceremony, X is O, R a' is Cl or optionally substituted C 1~30 Alkoxy (e.g., -(CH22) n CH3, where n is 1 to 21, for example, 1, 16, or -(CH22) m -NH2, where m is 2 to 10, for example 3 or 6, or -(CH22) p -OMe, where p is 1 to 21, for example 1 or 2; R b may be substituted C 1~6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl); R c is the bond or hydroxyl at the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R d is the bond to the internucleoside linkage to the preceding nucleoside.

[0031] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (I) is a nucleoside of Formula (IA) or (IB): During the ceremony, X is O, R a' is a halogen (e.g., F, Br, or Cl), R b is a halogen (e.g., F, Br, or Cl), R c is the bond or hydroxyl at the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and Rd is the bond to the internucleoside linkage to the preceding nucleoside.

[0032] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (I) is a nucleoside of Formula (IA) or (IB): During the ceremony, X is O, R a' is a halogen (e.g., F, Br, or Cl), hydroxyl, optionally substituted C 1~30 Alkoxy (e.g., -(CH22) n CH3, where n is 1 to 21, for example, 1, 16, or -(CH22) m -NH2, where m is 2 to 10, for example 3 or 6, or -(CH22) p -OMe, where p is 1 to 21, e.g., 1 or 2), or a bond to an internucleoside linkage to a subsequent nucleoside; R b may be substituted C 1~6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl) or halogen (e.g., F, Br, or Cl); R c is the bond or hydroxyl to the internucleoside linkage to the subsequent nucleoside, provided that R a' and R c only one of which is the bond to the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R d is the bond to the internucleoside linkage to the preceding nucleoside.

[0033] In some embodiments of any one of the aspects described herein, the antisense strand can be about 17 to 42 nucleotides in length. For example, the antisense strand is at least about 17, e.g., about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In some embodiments of any one of the aspects described herein, the antisense strand is about 19, about 20, about 21, about 22, about 23, about 24, about 25, or about 26 nucleotides in length. For example, the antisense strand is about 22, about 23, about 24, or about 25 nucleotides in length.

[0034] In some embodiments of any one of the aspects described herein, the sense strand can be about 15 to 40 nucleotides in length. For example, the sense strand is at least about 15, about 16, e.g., about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26 nucleotides in length, or longer. In some embodiments of any one of the aspects described herein, the sense strand is about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length. For example, the sense strand is about 21 nucleotides in length.

[0035] In some embodiments of any one of the aspects described herein, the sense strand is 15 nucleotides in length and the antisense strand is 18, 19, 20, 21, or 22 (e.g., 20) nucleotides in length. In some embodiments of any one of the aspects described herein, the sense strand is 19 nucleotides in length and the antisense strand is 19, 20, or 21 nucleotides in length. In some embodiments of any one of the aspects described herein, the sense strand is 20 nucleotides in length and the antisense strand is 20, 21, or 22 nucleotides in length. In some embodiments of any one of the aspects described herein, the sense strand is 21 nucleotides in length and the antisense strand is 21, 22, or 23 nucleotides in length. In some embodiments of any one of the aspects described herein, the sense strand is 20-24 (e.g., 22) nucleotides in length and the antisense strand is 34-38 (e.g., 36) nucleotides in length.

[0036] In some embodiments of any one of the aspects described herein, the sense strand is 21 nucleotides in length and the antisense strand is 22, 23, or 25 nucleotides in length.

[0037] In some embodiments of the various aspects described herein, the double-stranded region of the double-stranded nucleic acid (e.g., dsRNA) can be at least about 18, e.g., about 19, about 20, about 21, about 22, about 23, about 24, about 25 or more base pairs, e.g., about 21 base pairs.

[0038] In some embodiments of any one of the aspects described herein, the antisense strand is about 21, about 22, about 23, about 24, or about 25 nucleotides in length, the sense strand is about 21 nucleotides in length, and the dsRNA comprises a double-stranded region of at least 18, e.g., 19, 20, or 21 base pairs, e.g., 21 base pairs.

[0039] A double-stranded nucleic acid (e.g., dsRNA) can have blunt ends and / or single-stranded overhangs at its ends. For example, a double-stranded nucleic acid (e.g., dsRNA) can have a blunt end at the 5'-end of the antisense strand. In another example, a double-stranded nucleic acid (e.g., dsRNA) can have a single-stranded overhang of 1 to 5 (e.g., 1 or 2) nucleotides at the 3'-end of the antisense strand, for example, the 3'-end of the antisense strand extends beyond the 5'-end of the sense strand.

[0040] In some embodiments of any one of the aspects described herein, the double-stranded nucleic acid (e.g., dsRNA) comprises a blunt end at the 5'-end of the antisense strand and a single-stranded overhang of 1 to 5 (e.g., 1 or 2) nucleotides at the 3'-end of the antisense strand.

[0041] In some embodiments of any one of the aspects described herein, the double-stranded nucleic acid (e.g., dsRNA) comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleoside linkages. For example, the double-stranded nucleic acid (e.g., dsRNA) comprises at least four phosphorothioate internucleoside linkages, e.g., at least six phosphorothioate internucleoside linkages, or at least eight phosphorothioate internucleoside linkages.

[0042] It should be noted that phosphorothioate internucleoside linkages can be present in one or both strands. Furthermore, phosphorothioate internucleoside linkages can be present anywhere in the strand. For example, phosphorothioate internucleoside linkages can be present at one end of a strand, at both ends of a strand, and at an internal position within a strand, or at both ends and an internal position within a strand. Preferably, phosphorothioate internucleoside linkages are present at both ends of a strand.

[0043] In some embodiments, the antisense strand comprises at least one, e.g., two, three, four, or more, phosphorothioate internucleoside linkage. For example, the antisense strand comprises four or more phosphorothioate internucleoside linkages. In some embodiments of any one of the aspects described herein, the antisense strand comprises a phosphorothioate internucleoside linkage between positions 1 and 2, counting from the 3'-end of the strand, and a phosphorothioate internucleoside linkage between positions 1 and 2, counting from the 5'-end of the strand. In some further embodiments of any one of the aspects described herein, the antisense strand comprises a phosphorothioate internucleoside linkage between positions 1 and 2 and between positions 2 and 3, counting from the 3'-end of the strand, and a phosphorothioate internucleoside linkage between positions 1 and 2, counting from the 5'-end of the strand. In some further embodiments of any one of the aspects described herein, the antisense strand comprises phosphorothioate internucleoside linkages between positions 1 and 2 and between positions 2 and 3, counting from the 3' end of the strand, and between positions 1 and 2 and between positions 2 and 3, counting from the 5' end of the strand. In some further embodiments of any one of the aspects described herein, the antisense strand comprises phosphorothioate internucleoside linkages between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, counting from the 3' end of the strand, and between positions 1 and 2, counting from the 5' end of the strand. In some embodiments of any one of the aspects described herein, the antisense strand comprises a phosphorothioate internucleoside linkage between positions 1 and 2, counting from the 3' end of the strand, and between positions 1 and 2 and between positions 2 and 3, counting from the 5' end of the strand. In other further embodiments of any one of the aspects described herein, the antisense strand comprises a phosphorothioate internucleoside linkage between positions 1 and 2, counting from the 3' end of the strand, and between positions 1 and 2, 2 and 3, and 3 and 4, counting from the 5' end of the strand.

[0044] Like the antisense strand, the sense strand can also include one or more, e.g., two, three, four, or more, phosphorothioate internucleoside linkages. For example, the sense strand includes a phosphorothioate internucleoside linkage between positions 1 and 2, counting from the 5' end of the strand. In some embodiments of any one of the aspects described herein, the sense strand includes a phosphorothioate internucleoside linkage between positions 1 and 2, counting from the 5' end of the strand, and between positions 1 and 2, counting from the 3' end of the strand.

[0045] In some further embodiments of any one of the aspects described herein, the sense strand comprises phosphorothioate internucleoside linkages between positions 1 and 2 and between positions 2 and 3, counting from the 5' end of the strand. For example, the sense strand comprises phosphorothioate internucleoside linkages between positions 1 and 2 and between positions 2 and 3, counting from the 5' end of the strand, and between positions 1 and 2 and between positions 2 and 3, counting from the 3' end of the strand.

[0046] In some embodiments of any one of the aspects described herein, the double-stranded nucleic acid (e.g., dsRNA) comprises a ligand. For example, the sense strand comprises a ligand linked thereto. Note that the ligand can be linked to any available position, such as the 3'-terminal nucleotide, i.e., the nucleotide at position 1 (counting from the 3'-terminal), or the 5'-terminal nucleotide, i.e., the nucleotide at position 1 (counting from the 5'-terminal).

[0047]

[0013] Embodiments of the various aspects described herein include a ligand. Note that the ligand can be selected from the group consisting of peptides, centrins, antibodies (e.g., anti-CD4 antibodies and anti-CD117 antibodies), antibody fragments, T cell targeting ligands, B cell targeting ligands, cancer cell targeting ligands (e.g., DUPA, folate, and RGD), spleen targeting functionality, lung targeting functionality, bone marrow targeting functionality, phage display peptides, cell-penetrating peptides (CPPs), integrin ligands, polyanionic ligands, polycationic ligands, monovalent and polyvalent carbohydrates (e.g., GalNAc, mannose, mannose-6 phosphate, mucose, and mulucose), kidney targeting ligands, BBB-penetrating ligands, lipids, and amino acids (e.g., L-amino acids, D-amino acids, and β-amino acids). In some embodiments of any one of the aspects described herein, the ligand is monovalent or polyvalent N-acetylgalactosamine (GalNAc).

[0048] It should be noted that the double-stranded nucleic acid (e.g., dsRNA) described herein can comprise one or more additional nucleic acid modifications, such as nucleobase modifications, sugar modifications, inter-sugar linkage modifications, or any combination thereof.Thus, in some embodiments of any one of the aspects described herein, the double-stranded nucleic acid (e.g., dsRNA) comprises at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 2'-fluoro nucleotides.For example, the antisense strand and / or the sense strand independently comprise at least one, for example, 2, 3, 4, 5 or more, 2'-fluoro nucleotides.

[0049] In some embodiments of any one of the aspects described herein, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense strand. For example, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 14, and 16, counting from the 5' end of the antisense strand. In another non-limiting example, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 9, 14, and 16, counting from the 5' end of the antisense strand. In some further examples, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the antisense strand.

[0050] In some embodiments of any one of the aspects described herein, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand.

[0051] In some embodiments of any one of the aspects described herein, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, and 11, counting from the 5' end of the sense strand, or at positions 11, 13, and 15, counting from the 3' end of the sense strand. For example, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, 10, and 11, counting from the 5' end of the sense strand, or at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand.

[0052] In some embodiments of any one of the aspects described herein, the sense strand comprises 2'-fluoro nucleotides at positions 9, 10, and 11, counting from the 5' end of the sense strand, or at positions 11, 12, and 13, counting from the 3' end of the sense strand.

[0053] In some embodiments of any one of the aspects described herein, the antisense strand comprises 2'-fluoro nucleotides at least at positions 2, 14, and 16, counting from the 5' end of the antisense strand, and the sense strand comprises 2'-fluoro nucleotides at least at positions 7, 9, and 11, counting from the 5' end of the sense strand, or at least at positions 11, 13, and 15, counting from the 3' end of the sense strand. For example, the antisense strand comprises 2'-fluoro nucleotides at least at positions 2, 6, 14, and 16, counting from the 5' end of the antisense strand, and the sense strand comprises 2'-fluoro nucleotides at least at positions 7, 9, and 11, counting from the 5' end of the sense strand, or at least at positions 11, 13, and 15, counting from the 3' end of the sense strand. In another example, the antisense strand contains 2'-fluoro nucleotides at least at positions 2, 6, 9, 14, and 16, counting from the 5' end of the antisense strand, and the sense strand contains 2'-fluoro nucleotides at least at positions 7, 9, and 11, counting from the 5' end of the sense strand, or at least at positions 11, 13, and 15, counting from the 3' end of the sense strand. In yet another example, the antisense strand contains 2'-fluoro nucleotides at least at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the antisense strand, and the sense strand contains 2'-fluoro nucleotides at least at positions 7, 9, and 11, counting from the 5' end of the sense strand, or at least at positions 11, 13, and 15, counting from the 3' end of the sense strand.

[0054] In some further non-limiting examples, the antisense strand contains 2'-fluoro nucleotides at least at positions 2, 14, and 16, counting from the 5' end of the antisense strand, and the sense strand contains 2'-fluoro nucleotides at least at positions 7, 9, and 11, counting from the 5' end of the sense strand, or at least at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand. For example, the antisense strand contains 2'-fluoro nucleotides at least at positions 2, 6, 14, and 16, counting from the 5' end of the antisense strand, and the sense strand contains 2'-fluoro nucleotides at least at positions 7, 9, 10, and 11, counting from the 5' end of the sense strand, or at least at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand. In another example, the antisense strand contains 2'-fluoro nucleotides at least at positions 2, 6, 9, 14, and 16, counting from the 5' end of the antisense strand, and the sense strand contains 2'-fluoro nucleotides at least at positions 7, 9, 10, and 11, counting from the 5' end of the sense strand, or at least at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand. In yet another example, the antisense strand contains 2'-fluoro nucleotides at least at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the antisense strand, and the sense strand contains 2'-fluoro nucleotides at least at positions 7, 9, 10, and 11, counting from the 5' end of the sense strand, or at least at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand.

[0055] The dsRNAs described herein can contain one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, 2'-deoxy (i.e., 2'-H or DNA) nucleotides. For example, the antisense strand and / or the sense strand independently contain at least one, for example, 2, 3, 4, 5, or more, 2'-deoxy (i.e., 2'-H or DNA) nucleotide.

[0056] In some embodiments of any one of the aspects described herein, the antisense strand comprises DNA nucleotides at positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand. In some embodiments of any one of the aspects described herein, the antisense strand comprises DNA nucleotides at positions 2, 5, 7, 12, and 14, counting from the 5' end of the antisense strand. In some embodiments of any one of the aspects described herein, the antisense strand comprises DNA nucleotides at positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand.

[0057] In some embodiments of any one of the aspects described herein, the antisense strand comprises DNA nucleotides at positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand, and a 2'-fluoro nucleotide at position 14 of the antisense strand.

[0058] dsRNA described herein can comprise one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 2'-OMe nucleotides.For example, antisense strand and / or sense strand independently comprise at least one, for example, 2, 3, 4, 5 or more, 2'-OMe nucleotides.In some embodiments of any one of the aspects described herein, the remaining nucleotides in antisense strand, i.e., all except for the modifications specified herein, are 2'-OMe nucleotides.Similarly, in some embodiments of any one of the aspects described herein, the remaining nucleotides in antisense strand, i.e., all except for the modifications specified herein, are 2'-OMe nucleotides.

[0059] In some embodiments of any one of the aspects described herein, the antisense strand comprises a phosphate group or a phosphate analog or derivative thereof at its 5'-end. For example, the antisense strand comprises a 5'-vinylphosphonate nucleotide at its 5'-end. For example, the antisense strand comprises a 5'-E-vinylphosphate nucleotide at its 5'-end.

[0060] In some embodiments of any one of the aspects described herein, the dsRNA comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, locked nucleic acid (LNA) or bridged nucleic acid (BNA) nucleotide. For example, the antisense and / or sense strands independently comprise at least one, e.g., 2, 3, 4, 5, or more, LNA or BNA nucleotide.

[0061] In some embodiments of any one of the aspects described herein, the dsRNA comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, cyclohexene nucleic acid (CeNA) nucleotide. For example, the antisense and / or sense strands independently comprise at least one, e.g., 2, 3, 4, 5 or more, CeNA nucleotide.

[0062] In some embodiments of any one of the aspects described herein, the dsRNA comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, thermostabilizing modification. For example, the antisense and / or sense strands independently comprise at least one, e.g., 2, 3, 4, 5, or more, thermostabilizing modification.

[0063] In some embodiments of any one of the aspects described herein, the dsRNA comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, abasic nucleotide. For example, the antisense and / or sense strands independently comprise at least one, e.g., 2, 3, 4, 5, or more, abasic nucleotide.

[0064] In some embodiments of any one of the aspects described herein, dsRNA comprises at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 2'-deoxynucleotide.For example, antisense and / or sense strands independently comprise at least one, for example, 2, 3, 4, 5 or more, 2'-deoxynucleotide.In some embodiments, antisense strand comprises one or more, for example, 1, 2 or more, 2'-deoxynucleotide in single-stranded overhang.

[0065] In some embodiments of any one of the aspects described herein, the dsRNA comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, acyclic (e.g., unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or (S)-glycol nucleic acid (S-GNA)) nucleotide. For example, the antisense and / or sense strands independently comprise at least one, e.g., 2, 3, 4, 5, or more, UNA and / or GNA nucleotide.

[0066] In some embodiments of any one of the aspects described herein, the dsRNA comprises at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, thermodestabilizing modifications.For example, the antisense and / or sense strands independently comprise at least one, for example, 2, 3, 4, 5 or more, thermodestabilizing modifications.Exemplary thermodestabilizing modifications include, but are not limited to, abasic nucleotides, 2'-deoxynucleotides, acyclic nucleotides (for example, UNA, GNA and (S)-GNA), 2'-5' linked nucleotides (3'-RNA), threose nucleotides (TNA), 2' gem Me / F nucleotides, and mismatches with the opposite nucleotide in the other strand.

[0067] In some embodiments of any one of the aspects described herein, the antisense strand comprises at least one thermodestabilizing modification in the seed region of the antisense strand (i.e., positions 2-9 from the 5' end). For example, the antisense strand comprises a thermodestabilizing modification at least at one of positions 6, 7, or 8, counting from the 5' end of the strand. In some embodiments of any one of the aspects described herein, the antisense strand comprises a thermodestabilizing modification at position 7, counting from the 5' end of the strand.

[0068] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein include only 2'-geminally substituted nucleotides of Formula (I) and (II).

[0069] In some embodiments of any one of the aspects described herein, the oligonucleotide comprises only 2'-geminally substituted nucleotides of Formulas (I) and (II), and the oligonucleotide further comprises a ligand, e.g., monovalent or polyvalent N-acetylgalactosamine (GalNac), linked to the oligonucleotide. For example, the oligonucleotide comprises only 2'-geminally substituted nucleotides of Formulas (I) and (II), and the oligonucleotide further comprises a ligand, e.g., monovalent or polyvalent N-acetylgalactosamine (GalNac), linked to its 3' end.

[0070] In yet another aspect, provided herein are 2'-geminal substituted nucleotides or monomers of formula (III) or (III'): TIFF2024539093000012.tif38128 is provided.

[0071] In formulas (III) and (III'), X is O, S, C(R X )2, or N(R XN ) and Each R X are independently hydrogen, halogen, or optionally substituted C 1~4 Alkyl, C 1~4Haloalkyl, optionally substituted C 2~4 Alkenyl, or optionally substituted C 2~4 alkynyl, or both R X Overall, =O, =S, =N(R N ), or =CH2, R XN is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 1~30 alkyl-COH, or a nitrogen protecting group, B is an optionally modified nucleobase; R a is hydrogen, halogen, -OR a2 , -SR a3 , optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 Alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH2CH2O) m CH2CH2OR a4 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH2CH2NH) n CH2CH2-R a5 , NHC(O)R a4 a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, a solid support, a linker covalently attached to a solid support, or a reactive phosphorus group; R a2 is a hydrogen or hydroxyl protecting group, Ra3 is a hydrogen or sulfur protecting group, R a4 are independently generated for each occurrence, H, C1 to C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R a5 and R a5 is independently at each occurrence amino (NH), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino; m is 1 to 50; n is 1 to 50; R b may be substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 alkynyl, or halogen; R 3 is hydrogen, halogen, -OR c2 , -SR c3 , optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 Alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH2CH2O) r CH2CH2OR c4 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH2CH2NH) s CH2CH2-R c5 , NHC(O)R c4 a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, a solid support, a linker covalently attached to a solid support, or a reactive phosphorus group; Rc2 is a hydrogen or hydroxyl protecting group, R c3 is a hydrogen or sulfur protecting group, R c4 are independently generated for each occurrence, H, C1 to C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R c5 and R c5 is independently at each occurrence amino (NH), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino; r is 1 to 50; s is 1 to 50; R 4 is hydrogen, optionally substituted C 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, or optionally substituted C 1~6 Is it an alkoxy? Alternatively, R 4 and R a is, as a whole, 4'-C(R a11 R a12 ) v -Y-2' or 4'-YC(R a11 R a12 ) v -2', Y is -O-, -CH2-, -CH(Me)-, -C(CH3)2-, -S-, -N(R a13 )-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -OC(O)-, -C(O)O-, -N(R a13 )C(O)-, or -C(O)N(R a13 )- and R a11 and R a12 are independently H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl; R a13is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 1~30 alkyl-COH, or a nitrogen protecting group, v is 1, 2 or 3, Alternatively, R 4 and R c C, together with the atoms to which they are attached, may be substituted 3~8 Cycloalkyl, optionally substituted C 3~8 forming a cycloalkenyl or an optionally substituted 3- to 8-membered heterocyclyl, R 5 may be substituted C 1~6 Alkyl-R 5a , optionally substituted -C 2~6 Alkenyl-R 5a , or optionally substituted -C 2~6 Alkynyl-R 5a and R 5a -OR 5b , -SR 5c , hydrogen, a phosphorus group, a phosphorous group, a solid support, or a linker to a solid support, with the proviso that R 3a , R 3 and R 5 only one of which is a link to a solid support, R 5b is H or a hydroxyl protecting group, and R 5c is H or a sulfur protecting group.

[0072] In some embodiments of any one of the aspects described herein, the compound of Formula (III) is a compound of Formula (IIIA): TIFF2024539093000013.tif28128.

[0073] In some embodiments of any one of the aspects described herein, the compound of Formula (III) is a compound of Formula (IIIB'): The file is TIFF2024539093000014.tif33128.

[0074] In some embodiments of any one of the aspects described herein, the compound of Formula (III') is a compound of Formula (IIIA'): The file is TIFF2024539093000015.tif29128.

[0075] In some embodiments of any one of the aspects described herein, the compound of Formula (III') is a compound of Formula (IIIB'): TIFF2024539093000016.tif29128.

[0076] In some embodiments of any one of the aspects described herein, the compound of Formula (III) is a compound of Formula (IIIA) or (IIIB), wherein: X is O, R a is a halogen (e.g., F or Cl), hydroxyl, protected hydroxyl, optionally substituted C 1~30 Alkoxy (e.g., -(CH22) n CH3, where n is 1 to 21, for example, 1, 16, or -(CH22) m -NH2, where m is 2 to 10, for example 3 or 6, or -(CH22) p -OMe, where p is 1 to 21, e.g., 1 or 2), a reactive phosphorus(III) group (e.g., -OP(OR P )N(R P2 )2 (e.g., -OP(OCH2CH2CN)N(iPr)2), -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2)2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 ), a solid support, or a linker covalently attached to a solid support; R b may be substituted C 1~6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl); R 4 is hydrogen, R 3 is a reactive phosphorus(III) group (e.g., -OP(OR P )N(R P2 )2 (e.g., -OP(OCH2CH2CN)N(iPr)2), -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 ), a solid support, a linker covalently attached to the solid support, a hydroxyl, or a protected hydroxyl, provided that R a and R 3is a reactive phosphorus(III) group, a solid support, or a linker covalently attached to a solid support, and R 5 is -CH=CHR 5a where R 5a -P(O)(OR 5e )2, and each R 5e are independently hydrogen or optionally substituted C 1~30 It is alkyl.

[0077] In some embodiments of any one of the aspects described herein, the compound of Formula (III) is a compound of Formula (IIIA) or (IIIB), wherein: X is O, R a is F, Cl or optionally substituted C 1~30 Alkoxy (e.g., -(CH22) n CH3, where n is 1 to 21, for example, 1, 16, or -(CH22) m -NH2, where m is 2 to 10, for example 3 or 6, or -(CH22) p -OMe, where p is 1 to 21, for example 1 or 2; R b may be substituted C 1~6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl); R 4 is hydrogen, R 3 is a reactive phosphorus(III) group (e.g., -OP(OR P )N(R P2 )2 (e.g., -OP(OCH2CH2CN)N(iPr)2), -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SRP )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 ), a solid support, a linker covalently attached to the solid support, a hydroxyl, or a protected hydroxyl; and R 5 is -CH=CHR 5a where R 5a -P(O)(OR 5e )2, and each R 5e are independently hydrogen or optionally substituted C 1~30 It is alkyl.

[0078] In some embodiments of any one of the aspects described herein, the compound of Formula (III) is a compound of Formula (IIIA) or (IIIB), wherein: X is O, R a is Cl or optionally substituted C 1~30 Alkoxy (e.g., -(CH22) n CH3, where n is 1 to 21, for example, 1, 16, or -(CH22) m -NH2, where m is 2 to 10, for example 3 or 6, or -(CH22) p -OMe, where p is 1 to 21, for example 1 or 2; R b may be substituted C 1~6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl); R 4 is hydrogen, R 3is a reactive phosphorus(III) group (e.g., -OP(OR P )N(R P2 )2 (e.g., -OP(OCH2CH2CN)N(iPr)2), -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 ), a solid support, a linker covalently attached to the solid support, a hydroxyl, or a protected hydroxyl; and R 5 is -CH2OR 5b or -CH=CHR 5a where R 5b is H or a hydroxyl protecting group, and 5a -P(O)(OR 5e )2, where each R 5e are independently hydrogen or optionally substituted C 1~30 It is alkyl.

[0079] In some embodiments of any one of the aspects described herein, the compound of Formula (III) is a compound of Formula (IIIA) or (IIIB), wherein: X is O, R a is a halogen (e.g., Cl, Br, or F), R b is a halogen (e.g., Cl, Br, or F), R 4 is hydrogen, R3 is a reactive phosphorus(III) group (e.g., -OP(OR P )N(R P2 )2 (e.g., -OP(OCH2CH2CN)N(iPr)2), -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 ), a solid support, a linker covalently attached to the solid support, a hydroxyl, or a protected hydroxyl; and R 5 is -CH2OR 5b or -CH=CHR 5a where R 5b is H or a hydroxyl protecting group, and 5a -P(O)(OR 5e )2, where each R 5e are independently hydrogen or optionally substituted C 1~30 is alkyl, However, R 5 Ga-CH2OR 5b If (a) nucleobase B is not uracil, or (b) R a and R b cannot both be F.

[0080] In some embodiments of any one of the aspects described herein, the compound of Formula (III') is a compound of Formula (IIIA') or (IIIB'), wherein: X is O, Ra is a halogen (e.g., F, Br, or Cl), hydroxyl, protected hydroxyl, optionally substituted C 1~30 Alkoxy (e.g., -(CH22) n CH3, where n is 1 to 21, for example, 1, 16, or -(CH22) m -NH2, where m is 2 to 10, for example 3 or 6, or -(CH22) p -OMe, where p is 1 to 21, e.g., 1 or 2), a reactive phosphorus(III) group (e.g., -OP(OR P )N(R P2 )2 (e.g., -OP(OCH2CH2CN)N(iPr)2), -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 ), a solid support, a linker covalently attached to the solid support; R b may be substituted C 1~6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl), or halogen (e.g., F, Br, or Cl); R 4 is hydrogen, R 3 is a reactive phosphorus(III) group (e.g., -OP(OR P )N(R P2)2 (e.g., -OP(OCH2CH2CN)N(iPr)2), -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 ), a solid support, a linker covalently attached to the solid support, a hydroxyl, or a protected hydroxyl, provided that R a and R 3 is a reactive phosphorus(III) group, a solid support, or a linker covalently attached to a solid support, and R 5 is -CH2OR 5b or -CH=CHR 5a where R 5b is H or a hydroxyl protecting group, and 5a -P(O)(OR 5e )2, where each R 5e are independently hydrogen or optionally substituted C 1~30 It is alkyl.

[0081] In another aspect, provided herein is a method for inhibiting or reducing expression of a target gene in a subject, the method comprising administering to the subject (i) a double-stranded nucleic acid (e.g., dsRNA) described herein, wherein a first strand is complementary to the target gene, and / or (ii) an oligonucleotide described herein, wherein the oligonucleotide is complementary to the target gene. [Brief explanation of the drawings]

[0082] [Figure 1] FIG. 1 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 2] FIG. 2 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 3] FIG. 3 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 4] FIG. 4 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 5] FIG. 5 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 6] FIG. 6 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 7] FIG. 7 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 8] FIG. 8 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 9] FIG. 9 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 10] FIG. 10 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 11] FIG. 11 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 12] FIG. 12 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 13] FIG. 13 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 14] FIG. 14 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 15] FIG. 15 is a synthetic scheme for some exemplary compounds of formula (III). [Figure 16]Figure 16 shows the structures of the antiviral HCV drug sofosbuvir and the active metabolites that inspired the modified 2'-F / Me uridine (UF / Me) and cytidine (CF / Me) and corresponding 5'-vinylphosphonate isomers (E-VP-UF / Me and Z-VP-UF / Me) studied here. [Figure 17] Figures 17A-17D show the in vitro potency of fully 2'-modified siRNAs targeting (Figure 17A) Ttr, (Figure 17B) Pten, and (Figure 17C) F7. See Table 8 for experimental conditions. 2'-F and 2'-OMe nucleotides are represented as green or black circles, respectively. Yellow bars represent PS linkages, where VP is 5'-(E)-vinylphosphonate and zVP is 5'-(Z)-vinylphosphonate. Error bars indicate standard deviation from the mean. [Figure 18]Figures 18A and 18B show the reduction of seed-mediated off-target activity by incorporation into AS7. Figure 18A) On-target and off-target effects were assessed using a dual-luciferase reporter assay. A luciferase reporter plasmid was co-transfected with the indicated siRNA into COS-7 cells. Cells were harvested 48 hours later, and luciferase activity was assayed. Target survival was calculated by dividing the ratio of Renilla luciferase to firefly luciferase signals at each siRNA concentration by the ratio in the absence of siRNA. Figure 18B) Transcriptional dysregulation in primary rat hepatocytes. Primary rat hepatocytes were transfected with 50 nM of the indicated siRNA. The upper panel shows the parent (si19) and the lower panel shows the modified (si20) variants. After 48 hours, total RNA was isolated for RNA-seq analysis. Dots represent individual rat transcripts, their average read counts, and the level of expression change compared to mock-transfected controls. Gray dots represent genes that were not differentially expressed after siRNA treatment compared to controls, while blue and red dots represent differentially expressed genes (false positive rate <0.05) with or without canonical miRNA matches (8-mer, 7-mer-A1, 7-mer-m8) to the seed region, respectively. On-target knockdown of Ttr is indicated by circled dots. Log2 fold change and cumulative distribution plots are shown on the left and right, respectively. [Figure 19]Figures 19A-19D show the effect of 2'-F / Me modifications on in vivo activity. Figures 19A and 19B) C57BL / 6 mice (n=3) were administered a single dose of 1 mg / kg (pink) or 3 mg / kg (gray) of Figure 19A) F7-targeting siRNA intravenously in an LNP formulation or a single dose of Figure 1B) F7-targeting GalNAc-conjugated siRNA subcutaneously. Control animals received PBS. Serum F7 protein levels were measured at the parent nadir: 48 hours for the LNP formulation and 10 days for the GalNAc-conjugated siRNA. Figures 19C and 19D) C57BL / 6 mice (n=3) were administered a single dose of 1 mg / kg of the indicated TTR-targeting siRNA, and serum protein levels were monitored for up to 28 days. 2'-F, 2'-OMe, deoxyribonucleotides, and ribonucleotides are represented by green, black, blue, and red circles, respectively. Yellow bars represent PS junctions. Data points are normalized to pre-treatment F7 or TTR levels, and values ​​are group means ± SD. [Figure 20] Figure 20A illustrates the steric clash that occurs as a result of the introduction of a 2'-β-C-methyl group into a single nucleotide in a 2'-F-modified RNA A-form duplex (PDB ID 3P4A). Figure 20B shows that after energy minimization by molecular mechanics, there is no clash between the methyl group and its nearest neighbor, but stacking between uridines is lost. The carbon and hydrogen atoms of the methyl are colored yellow and white, respectively, and the fluorine atoms are light green. Short contacts are indicated by arrows. Watson-Crick hydrogen bonds and other selected distances are shown as thin solid lines, and the backbone torsion angle range is depicted in Figure 20A. [Figure 21]Figures 21A-21D show the modeled conformations of 2'-F / Me nucleotides incorporated into the siRNA guide strand bound to human Ago2 at (Figure 21A) position 1, (Figure 21B) position 2, (Figure 21C) position 6, and (Figure 21D) position 7 of the antisense strand. Methyl carbon and hydrogen atoms are colored yellow and white, respectively, and fluorine atoms are colored light green. Short contacts are indicated by arrows. The initial conformation of the antisense strand seen in the crystal structure of the human Ago2:miR-20a complex (PDB ID 4F3T)53 is indicated by a thin black line. Potentially favorable contacts are indicated by dashed lines in Figure 21D. [Figure 22] Figures 22A–C show models of VP-2'-F / Me-modified nucleotides in AS1 bound to the Ago2 MID domain. (Figure 22A) E-VP with a C2'-endosugar conformation; carbon atoms are colored purple. (Figure 22B) Z-VP with an O4'-endosugar conformation; carbon atoms are colored light blue. (Figure 22C) Superposition of E-VP and Z-VP nucleotides. The distance between the two phosphorus atoms (1.85 Å) is indicated by a double arrow. The VP moiety, 2'-F (light green), and 2'-Me carbon (yellow) are highlighted in ball-and-stick mode. Salt bridges and hydrogen bonds are drawn with thin solid lines, and selected Ago2 side chains are labeled. [Figure 23]Figures 23A and 23B show the source of improved resistance to exonuclease degradation by 2'-F / Me-modified oligonucleotides. (Figure 23A) Model of oligo(dT) (yellow carbons) with two 5'-terminal 2'-F / Me-U residues (cyan carbons) bound to the active site of D. melanogaster Xrn1 5'-exoribonuclease. (Figure 23B) Model of oligo(dT) (yellow carbons) with two 3'-terminal 2'-F / Me-U residues (cyan carbons) bound to the active site of E. coli DNA polymerase I Klenow fragment 3'-exonuclease. The distance between the 2'-Me carbon atom and the phosphorus atom is indicated by an orange arrow. The distance between the 2'-Me carbon and selected protein and DNA atoms is indicated by black arrows. The 2'-F (light green), 2'-Me carbon (yellow), phosphorus (orange), non-bridging phosphate oxygen (red), and metal ion are highlighted in stick-and-ball mode. Salt bridges and hydrogen bonds are drawn with thin solid lines, the metal ion coordination sphere is drawn with dashed lines, and selected Xrna1 and Klenow fragment side chains are labeled. All water molecules have been omitted except for those coordinated to the catalytic metal ion. [Figure 24] Figures 24A-24D are thermal denaturation (Tm) curves of the modified duplex (2.5 uM) in 6.8xPBS ([NaCl] = 931.6 mM, [KCl] = 18.4 mM, [Na2HPO4] = 68 mM, [KH2PO4] = 12.2 mM, pH 7.4). [Figure 25] Figures 25A-25D show curves of modified oligonucleotides in the presence of 3' exonuclease. Oligonucleotides (0.1 mg / mL) were incubated with 150 mU / mL SVPD in 50 mM Tris, pH 7.2, 10 mM MgCl2, and full-length products were monitored by IEX-HPLC. ON13(X) = dT19X-3', ON14(X) = dT18XdT, ON15(X) = dT18X2, ON16(X) = dT18X dT-3', ON17(X) = dT18 X-3', ON18(X) = dT18X X-3', PS linkage = . [Figure 26]Figures 26A-26D show degradation curves of modified oligonucleotides in the presence of 5' exonuclease. Oligonucleotides (0.1 mg / mL) were incubated with PD II (500 mU / mL) in 50 mM sodium acetate buffer (pH 6.5) containing 10 mM MgCl2 and monitored by IEX-HPLC. ON19(X) = 5'-XdT19, ON20(X) = 5'-dTXdT18, ON21(X) = 5'-X2dT18, ON16(X) = dT18X·dT-3', ON17(X) = dT18·X-3', ON18(X) = dT18X·X-3', PS linkage = . [Figure 27] Figure 27 shows the in vitro metabolic stability after 24 hours of incubation at 37°C in rat liver homogenate. Arrows represent the percentage of strands observed by LC-MS, with the tail direction representing the observed fragment. Green spheres = 2'-F nucleotides, black spheres = 2'-OMe nucleotides, and pink spheres = 2'-F / Me uridine, VP = 5'-(E)-vinylphosphonate, zVP = 5'-(Z)-vinylphosphonate. [Figure 28] FIG. 28 shows approximate dose-response curves for determining IC50 values ​​of siRNAs targeting TTR mRNA. [Figure 29] FIG. 29 is an approximate dose-response curve for determining IC50 values ​​of siRNAs targeting PTEN mRNA. [Figure 30] FIG. 30 is an approximate dose-response curve for determining IC50 values ​​of siRNAs targeting FVII mRNA. [Figure 31]Figure 31 shows the effect of 2'-F / Me modification on in vivo activity. ApoB-targeting siRNA was administered at 10 mg / kg, and relative mRNA expression of target genes was calculated by qPCR from liver tissue on day 7. 2'-F, 2'-OMe, deoxyribonucleotides, and ribonucleotides are represented as green, black, blue, and red circles, respectively. Yellow bars represent PS linkages, and VP is 5'-(E)-vinylphosphonate. Data points are normalized to pre-administration ApoB levels, and values ​​represent group means ± SD. [Figure 32] Figure 32 is a schematic diagram illustrating the source of POLG's instability for incorporating 2'-F / Me-modified nucleotides. The active site is shown in the crystal structure (PDB ID 4ZTZ) of the ternary POLG·DNA·dCTP Mg2+ complex. The view is of the minor groove of the duplex formed by the template (pink carbon atoms) and primer (cyan carbon atoms). The 2'-F / Me CMP (purple carbon atoms) is superimposed on the incoming dCTP (gold carbon atoms). Two Mg2+ ions are visible in the background as gray spheres. The distance of 2.78 Å between the 2'-F / Me methyl carbon (highlighted in yellow) and the center of mass of the Tyr-951 ring (black dot) indicates a short contact (the vdW radius of the methyl group, 2 Å, plus the vdW radius of the phenyl carbon, 1.5 Å, equals 3.5 Å). Selected distances are indicated by thin solid lines. [Figure 33A] Figures 33A and 33B are synthetic schemes for some exemplary compounds. [Figure 33B] See legend to Figure 33A. [Figure 34] Figure 34 is a schematic diagram of p-oligonucleotides for therapeutic utility using exemplary nucleoside building blocks. As shown, any number of building blocks can be assembled into an oligonucleotide and attached to a selected ligand (e.g., TriGalNAc). For example, 2'-gem Me / F compounds with cytosine nucleobases can be used for delivery to hepatocytes in the liver for HCV. Similarly, gemcitabine can be delivered to, for example, hepatocellular carcinoma. DETAILED DESCRIPTION OF THE INVENTION

[0083] Detailed Description It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of the invention as claimed. As used herein, the use of the singular includes the plural unless specifically stated otherwise. The use of "or" herein means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including" and other forms, such as "includes" and "included," is not limiting. Furthermore, terms such as "element" or "component" include both elements and components that include one unit and elements and components that include two or more subunits, unless specifically stated otherwise.

[0084] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. Any documents or portions of documents referred to in this application, including, for example, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for all purposes.

[0085] X In some embodiments of any one of the aspects described herein, X is O, S, C(R X )2, or N(R XN ) X is C(R X )2, then each R X are independently hydrogen, halogen, or optionally substituted C 1~4 Alkyl, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, or optionally substituted C 2~4 alkynyl, or both R X Overall, =O, =S, =N(R XN), or =CH2. For example, X is O. If X is N(R XN ), then R XN is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 1~30 alkyl-CO2H, or a nitrogen protecting group.

[0086] In some embodiments of any one of the aspects described herein, X is O.

[0087] Nucleic acid bases In some embodiments of any one of the aspects described herein, B is H or a nucleobase. Note that the nucleobase can be natural, unnatural and / or modified nucleobase. Exemplary natural nucleobases include, but are not limited to, adenine, cytosine, guanine, thymine and uracil. "Unnatural nucleobase" refers to a nucleobase other than adenine, guanine, cytosine, uracil or thymine.Exemplary unnatural nucleobases include inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, and substituted or modified analogs of adenine, guanine, cytosine, and uracil, such as 2-aminoadenine and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil, and 5-halocytosine. , 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine and 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkylcytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, Uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N 4

[0023] Additional purines and pyrimidines include, but are not limited to, acetylcytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanine, or O-alkylated bases. Additional purines and pyrimidines include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Concise Encyclopedia of Polymer Science and Engineering, edited by Kroschwitz, JI, John Wiley & Sons, 1990, pages 858-859, and those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, the contents of all of which are incorporated herein by reference.

[0088] In some embodiments, the unnatural nucleobase is inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(Isopentenyl)adenine, 7-(Deaza)adenine, 8-(Alkenyl)adenine, 8-(Alkyl)adenine, 8-(Alkynyl)adenine, 8-(Amino)adenine, 8-(Halo)adenine, 8-(Hydroxyl)adenine, 8-(Thioalkyl)adenine, 8-(Thiol)adenine, N 6 -(Isopentyl)adenine, N 6 -(methyl)adenine, N 6 ,N 6-(Dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine cytosine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4 -(Acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil , 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonylmethyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil Uracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1 ,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl (hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deazaino Indolyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl phenyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidines, N, 2 -substituted purines, N6 -substituted purines, O 6 -substituted purines, substituted 1,2,4-triazoles, and any O- or N-alkylated derivatives thereof.

[0089] In some embodiments, nucleobase is modified nucleobase.That is, nucleobase comprises nucleobase modification as described herein, for example, nucleobase is any substitution or modified analogue of natural nucleobase.The example of nucleobase modification includes: C-5 pyrimidine modification with alkyl group or amino alkyl and other cationic group, for example, guanidinium and amidine functional group, known in the art; N-terminal nucleotide of purine with alkyl group or amino alkyl and other cationic group, for example, guanidinium and amidine functional group, 2 - and N 6 -modifications, G-clamp, guanidinium G-clamp, and pseudouridine.

[0090] In some embodiments of any one of the above aspects, nucleobase is a universal nucleobase.The universal nucleobase referred to herein is any modified or unmodified natural or unnatural nucleobase that can base pair with adenine, cytosine, guanine and uracil without substantially affecting the melting behavior, the recognition by intracellular enzymes or the activity of the oligonucleotide that contains the universal nucleobase.Exemplary universal nucleobases include 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylimidazole, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, These include, but are not limited to, 9-methyl-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-propynylisocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof.

[0091] In some embodiments of any one of the aspects described herein, the nucleobase (e.g., B) is a protected nucleobase. As used herein, "protected nucleobase" refers to a nucleobase that includes a nitrogen-protecting group, an oxygen-protecting group, and / or a sulfur-protecting group.

[0092] In some embodiments of any one of the aspects described herein, the nucleobase (e.g., B) is a nucleobase selected from adenine, cytosine, guanine, thymine, uracil, and any modified, protected, or substituted analogue thereof.

[0093] R a' In some embodiments of any one of the aspects described herein, R a' is halogen, hydrogen, -OR a2 , -SR a3 , optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 Alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH2CH2O) m CH2CH2OR a4 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH2CH2NH) n CH2CH2-R a5 , NHC(O)R a4 , a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, a solid support, a linker covalently attached to a solid support, or an attachment to a subsequent internucleoside linkage to a nucleoside.

[0094] In some embodiments of any one of the aspects described herein, R a' is a halogen. For example, R a' is fluoro (F). In some examples, R a' is chloro (Cl).

[0095] In some embodiments of any one of the above aspects, R a' -OR a2 If R a2 can be hydrogen or a hydroxyl protecting group. For example, R a2 In some embodiments of any one of the aspects described herein, can be hydrogen.

[0096] R a'Ga-SR a3 If R a3 can be hydrogen or a sulfur protecting group. Thus, in some embodiments of any one of the above aspects, R a3 is hydrogen.

[0097] R a' -O(CH2CH2O) m CH2CH2OR a4 where m is 1 to 50, and R a4 are independently generated for each occurrence, H, C1 to C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R a5 and R a5 is independently at each occurrence amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0098] R a' -NH(CH2CH2NH) n CH2CH2-R a5 where n is 1 to 50, and R a5 is independently at each occurrence amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0099] In some embodiments of any one of the aspects described herein, R a' is hydrogen, halogen, -OR a2 or optionally substituted C1 to C 30 Alkoxy. For example, R a is halogen, -OR a2 or optionally substituted C1 to C 30 In some embodiments of any one of the aspects described herein, R a is F, Cl, OH or optionally substituted C1-C 30 It is an alkoxy.

[0100] In some embodiments of any one of the aspects described herein, R a is a halogen. For example, R a is fluoro (F). In some examples, R a is chloro (Cl).

[0101] In some embodiments of any one of the aspects described herein, R a' are OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkyl). koxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C1-C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 30 alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R a' is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, 30 In some embodiments of any one of the aspects described herein, R a' is -O(CH2) p CH3, where p is 1 to 21. For example, p is 14, 15, 16, 17, or 18. In one non-limiting example, p is 16.

[0102] In some embodiments of any one of the above aspects, R a' is -O(CH2) q R a7 where q is between 2 and 10, and R a7 is C1-C6 alkoxy, amino (NH2), CO2H, OH, or halo. For example, R a7 is —CH or —NH. Thus, in some embodiments of any one of the above aspects, R a is -O(CH2) q -OMe or R a is -O(CH2) q -NH2.

[0103] In some embodiments of any one of the aspects described herein, q is 2, 3, 4, 5, or 6. For example, q is 2, 3, or 6. In one non-limiting example, q is 2. In another non-limiting example, q is 3 or 6.

[0104] In some embodiments of any one of the aspects described herein, R a' is C1-C6 haloalkyl. For example, R a' is C1-C4 haloalkyl. In some embodiments of any one of the aspects described herein, R a is -CF3, -CF2CF3, -CF2CF2CF3 or -CF2(CF3)2.

[0105] In some embodiments of any one of the aspects described herein, R a' -OCH(CH2OR a8 )CH2OR a9 where R a8 and R a9 are independently H, optionally substituted C1 to C 30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R a8 and R a9are independently optionally substituted C1 to C 30 It is alkyl.

[0106] In some embodiments of any one of the aspects described herein, R a' -CH2C(O)NHR a10 where R a10 is H, optionally substituted C1 to C 30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R a10 is H or optionally substituted C1 to C 30 In some embodiments, R a10 is an optionally substituted C1-C6 alkyl.

[0107] In some embodiments of any one of the aspects described herein, R a' can be the bond to the internucleoside linkage to the subsequent nucleoside.

[0108] In some embodiments of any one of the aspects described herein, R a' can be a linker to a solid support.

[0109] R b In some embodiments of any one of the aspects described herein, R b may be substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 alkynyl, or halogen. For example, R bare OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkyl). koxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 1~30 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R b is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, 30 It is alkyl.

[0110] In some embodiments, R b is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, C 1~30 In some embodiments, R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl. For example, R b is methyl, vinyl, ethynyl, allyl, or propargyl. In some embodiments of any one of the aspects described herein, R b is methyl.

[0111] In some embodiments of any one of the aspects described herein, R a' is a halogen and R b is optionally substituted C 1~30 alkyl. For example, R a' is F and R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl. a' is F and R b is methyl.

[0112] In some embodiments of any one of the aspects described herein, R a is a halogen and R b is optionally substituted C 1~30 alkyl. For example, R a is F and R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl. a is F and R b is methyl.

[0113] In some embodiments of any one of the aspects described herein, R a' and R b is a halogen. For example, R a' and R b are independently F, Cl, Br or I. a' and R b Note that R can be the same or different. In some embodiments of any of the aspects described herein, R a' and R b is F. In some embodiments of any one of the aspects described herein, R a' and R b and cannot simultaneously be F. In some embodiments of any one of the aspects described herein, Ra' and R b is Cl or Br.

[0114] In some embodiments of any one of the aspects described herein, R a and R b is a halogen. For example, R a and R b are independently F, Cl, Br or I. a and R b Note that R can be the same or different. In some embodiments of any of the aspects described herein, R a and R b is F. In some embodiments of any one of the aspects described herein, R a and R b and cannot simultaneously be F. In some embodiments of any one of the aspects described herein, R a and R b is Cl or Br.

[0115] R c In some embodiments of any one of the aspects described herein, R c is the bond to the internucleoside linkage to the subsequent nucleoside, hydrogen, halogen, -OR c2 , -SR c3 , optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 Alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH2CH2O) r CH2CH2OR c4 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH2CH2NH) s CH2CH2-Rc5 , NHC(O)R c4 , a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, a solid support, or a linker covalently attached to a solid support.

[0116] In some embodiments of any one of the above aspects, R b and R c One of the bonds is the internucleoside linkage to the subsequent nucleoside. For example, R c is the bond to the internucleoside linkage to the subsequent nucleoside.

[0117] In some embodiments of any one of the above aspects, R c -OR c2 If R c2 can be hydrogen or a hydroxyl protecting group. For example, R c2 In some embodiments of any one of the aspects described herein, can be hydrogen.

[0118] R c Ga-SR c3 If R c3 can be hydrogen or a sulfur protecting group. Thus, in some embodiments of any one of the above aspects, R c3 is hydrogen.

[0119] R c -O(CH2CH2O) r CH2CH2OR c4 where r can be 1 to 50, and R c4 are independently generated for each occurrence, H, C1 to C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R c5 and R c5 is independently at each occurrence amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0120] R c But -NH(CH2CH2NH) s CH2CH2-R c5 where s can be 1 to 50, and R c5 can be, independently at each occurrence, amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0121] In some embodiments of any one of the aspects described herein, R c is hydrogen, halogen, -OR c2 or optionally substituted C1 to C 30 Alkoxy. For example, R c is halogen, -OR c2 or optionally substituted C1 to C 30 In some embodiments of any one of the aspects described herein, R c is F, OH or optionally substituted C1-C 30 It is an alkoxy.

[0122] In some embodiments of any one of the aspects described herein, R c are OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkyl). koxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p-OH, CH2-[CH(OH)] m -(CH2) p C1-C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 30 alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R c is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, 30 alkyl. For example, R c may be substituted with NH2 or C1-C6 alkoxy, 30 It is an alkoxy.

[0123] In some embodiments of any one of the aspects described herein, R c is -O(CH2) t CH3, where t is 1 to 21. For example, t is 14, 15, 16, 17, or 18. In one non-limiting example, t is 16.

[0124] In some embodiments of any one of the above aspects, R c is -O(CH2) u R c7 where u is 2 to 10, and R a7 is C1-C6 alkoxy, amino (NH2), CO2H, OH, or halo. For example, R c7 is —CH or NH. Thus, in some embodiments of any one of the aspects described herein, R c is -O(CH2) u -OMe or R c is -O(CH2) u It is NH2.

[0125] In some embodiments of any one of the aspects described herein, u is 2, 3, 4, 5, or 6. For example, u is 2, 3, or 6. In one non-limiting example, u is 2. In another non-limiting example, u is 3 or 6.

[0126] In some embodiments of any one of the aspects described herein, R c is C1-C6 haloalkyl. For example, R c is C1-C4 haloalkyl. In some embodiments of any one of the aspects described herein, R c is -CF3, -CF2CF3, -CF2CF2CF3 or -CF2(CF3)2.

[0127] In some embodiments of any one of the aspects described herein, R c -OCH(CH2OR c8 )CH2OR c8 where R c8 and R c9 are independently H, optionally substituted C1 to C 30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R c8 and R c9 are independently optionally substituted C1 to C 30 It is alkyl.

[0128] In some embodiments of any one of the aspects described herein, R c -CH2C(O)NHR c10 where R c10 is H, optionally substituted C1 to C 30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R c10 is H or optionally substituted C1 to C 30 In some embodiments, R a10 is an optionally substituted C1-C6 alkyl.

[0129] In some embodiments of any one of the aspects described herein, R cis a solid support or a linker covalently attached to a solid support.

[0130] In some embodiments of any one of the aspects described herein, R a' is a halogen and R b is optionally substituted C 1~30 alkyl, and R c is the bond to the internucleoside linkage to the subsequent nucleoside. For example, R a' is F and R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl; R c is the bond to the internucleoside linkage to the subsequent nucleoside. In some embodiments of any one of the aspects described herein, R a' is F and R b is methyl and R c is the bond to the internucleoside linkage to the subsequent nucleoside.

[0131] R 4 In some embodiments of any one of the aspects described herein, R 4 is hydrogen, optionally substituted C 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, or optionally substituted C 1~6 For example, R 4 can be hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl. For example, R b is methyl, vinyl, ethynyl, allyl or propargyl.

[0132] In some embodiments of any one of the aspects described herein, R 4 is H.

[0133] In some embodiments of any one of the aspects described herein, R a and R 4 is, as a whole, 4'-C(R a11 R a12 ) v -Y-2' or 4'-YC(R a11 R a12 ) v -2', and v is 1, 2, or 3, where Y is -O-, -CH2-, -CH(Me)-, -C(CH3)2-, -S-, -N(R a13 )-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -OC(O)-, -C(O)O-, -N(R a13 )C(O)-, or -C(O)N(R a13 )- and R a11 and R a12 are independently H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl, and R a13 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 1~30 alkyl-CO2H, or a nitrogen protecting group.

[0134] In some embodiments of any one of the above aspects, v is 1. In some other embodiments of any one of the above aspects, v is 2.

[0135] In some embodiments, Y is O. For example, R a and R 4 is, as a whole, 4'-C(R a11 R a12 ) v -O-2'.

[0136] R attached to the same carbon a11 and Ra12 Note that the can be the same or different. For example, R a11 and R a12 can be H, and one of R a11 and R a12 The other of R can be an optionally substituted C1-C6 alkyl. a11 and R a12 One of the groups can be H and the other can be OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl(s) i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p and C1-C6 alkyl optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R a11 and R a12 are independently H or C1-C optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy. 30 In some embodiments of any one of the above aspects, R a11 and R a12One of the groups is H, and the other is C1-C6 alkyl optionally substituted with C1-C6 alkoxy. For example, R a11 and R a12 One of the groups is H and the other is —CH3 or CH2OCH3.

[0137] In some embodiments of any one of the above aspects, R a11 and R a12 For example, R attached to the same C a11 and R a12 is H.

[0138] In some embodiments of any one of the above aspects, R a and R 4 is, as a whole, 4'-CH2-O-2', 4'-CH(CH3)-O-2', 4'-CH(CH2OCH3)-O-2', or 4'-CH2CH2-O-2'.

[0139] In some embodiments of any one of the aspects described herein, R 4 is H.

[0140] Or, in some embodiments of any one of the aspects described herein, R c and R 4 C, together with the atoms to which they are attached, may be substituted 3~8 Cycloalkyl, optionally substituted C 3~8 It forms a cycloalkenyl or an optionally substituted 3- to 8-membered heterocyclyl.

[0141] In some embodiments of any one of the aspects described herein, R a' is a halogen and R b is optionally substituted C 1~30 alkyl, and R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R 4 is H. For example, R a' is F and Rb is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl; R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R 4 is H. In some embodiments of any one of the aspects described herein, R a' is F and R b is methyl and R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R 4 is H.

[0142] R d In various embodiments described herein, R d -CH(R d1 )-R d2 or -C(R d1 )=CHR d2 where R d1 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted -C 2~30 alkenyl, or optionally substituted -C 2~30 alkynyl, and R d2 is the bond to the internucleoside linkage to the preceding nucleotide, and R d3 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 1~30 alkyl-CO2H, or a nitrogen protecting group.

[0143] In some embodiments of any one of the aspects described herein, X d is O or a bond. For example, X d is O.

[0144] In some embodiments of the various aspects described herein, R d -CH(R d1 )-X d -R d2 is.

[0145] In some embodiments of the various aspects described herein, R d -CH(R d1 )-X d -R d2 where R d1 is H or is OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1 ~C8 alkoxy), O(C1-C8) haloalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C1-C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 30 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R d1 is H. In some other non-limiting examples, R d1 is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, 30 It is alkyl.

[0146] In some embodiments of the various aspects described herein, R d -CH(R d1 )-OR d2 where R d1 is H or is OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1 ~C8 alkoxy), O(C1-C8) haloalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C1-C optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy; 30 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R d1 is H. In some other non-limiting examples, R d1 is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, 30 It is alkyl.

[0147] In some embodiments of any one of the aspects described herein, R d -CH2-OR d2 is.

[0148] In some embodiments of any one of the aspects described herein, R d -C(R d1 )=CHR d2 -C(R d1 )=CHR d2 Note that the double bond in can be in the cis or trans configuration. Thus, in some embodiments of any one of the above aspects, R d -C(R d1 )=CHR d2 wherein the double bond is in a cis configuration. In some other embodiments of any one of the above aspects, R d -C(R d1 )=CHR d2 wherein the double bond is in a trans configuration. In some embodiments of any one of the aspects described herein, R d is -CH=CHR d2 is.

[0149] In some embodiments of any one of the aspects described herein, R a' is a halogen and R b is optionally substituted C 1~30 alkyl, and R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R 4 is H and R d is the bond to the internucleoside linkage to the preceding nucleotide. For example, R a' is F and R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl; R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R 4 is H and R d is the bond to the internucleoside linkage to the preceding nucleotide. In some embodiments of any one of the aspects described herein, R a' is F and R b is methyl and R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R4 is H and R d is the bond to the internucleoside linkage to the preceding nucleotide.

[0150] In some embodiments of any one of the aspects described herein, R a' is a halogen and R b is optionally substituted C 1~30 alkyl, and R c is a hydroxyl, a solid support, or a linker covalently linked to a solid support, and R 4 is H and R d is the bond to the internucleoside linkage to the preceding nucleotide. For example, R a' is F and R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl; R c is a hydroxyl, a solid support, or a linker covalently linked to a solid support, and R 4 is H and R d is the bond to the internucleoside linkage to the preceding nucleotide. In some embodiments of any one of the aspects described herein, R a' is F and R b is methyl and R c is a hydroxyl, a solid support, or a linker covalently linked to a solid support, and R 4 is H and R d is the bond to the internucleoside linkage to the preceding nucleotide.

[0151] In some embodiments of any one of the aspects described herein, R a' is a halogen and R b is optionally substituted C 1~30 alkyl, and R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R 4 is H and R d -CH2-OR d2 For example, R a'is F and R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl; R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R 4 is H and R d -CH2-OR d2 In some embodiments of any one of the aspects described herein, R a' is F and R b is methyl and R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R 4 is H and R d -CH2-OR d2 is.

[0152] In some embodiments of any one of the aspects described herein, R a' is a halogen and R b is optionally substituted C 1~30 alkyl, and R c is a hydroxyl, a solid support, or a linker covalently linked to a solid support, and R 4 is H and R d -CH2-OR d2 For example, R a' is F and R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl; R c is a hydroxyl, a solid support, or a linker covalently linked to a solid support, and R 4 is H and R d -CH2-OR d2 In some embodiments of any one of the aspects described herein, R a' is F and R b is methyl and R c is a hydroxyl, a solid support, or a linker covalently linked to a solid support, and R 4 is H and R d-CH2-OR d2 is.

[0153] R e In various embodiments described herein, R e is optionally substituted -C 2~6 Alkenyl-R e1 , optionally substituted C 1~6 Alkyl-R e1 , or optionally substituted -C 2~6 Alkynyl-R e1 In various embodiments described herein, R e1 -OR e2 , -SR e3 , -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -P(S)(SR e5 )2, -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(O)(OR e4 )2, -SP(S)(OR e4 )2, -SP(S)(SR e5 )(OR e4 ), or -SP(S)(SR e5 )2, where R e2 is a hydrogen or oxygen protecting group, and R e3 is a hydrogen or sulfur protecting group, and each R e4 are independently hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 alkynyl, an oxygen protecting group, or an alkali metal or transition metal group with a total charge of +1, and each R e5 are independently hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30Alkenyl, or optionally substituted C 2~30 alkynyl, or sulfur protecting group. 2~6 Alkenyl-R e1 Note that the double bond in can be in the cis or trans configuration.

[0154] In some embodiments of any one of the above aspects, -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), SP(O)(OR e4 )2, -SP(S)(OR e4 )2, and -SP(S)(SR e5 )(OR e4 ) with at least one R e4 is hydrogen.

[0155] In some embodiments of any one of the above aspects, -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), SP(O)(OR e4 )2, -SP(S)(OR e4 )2, and -SP(S)(SR e5 )(OR e4 ) with at least one R e4 is an alkali or transition metal with a total charge of +1.

[0156] In some other embodiments of any one of the above aspects, -P(O)(OR e4 )2, -P(S)(OR e4)2, -P(S)(SR e5 )(OR e4 ), -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), SP(O)(OR e4 )2, -SP(S)(OR e4 )2, or -SP(S)(SR e5 )(OR e4 ) with at least one R e4 is not hydrogen. For example, P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), SP(O)(OR e4 )2, -SP(S)(OR e4 )2, and -SP(S)(SR e5 )(OR e4 ) at least one R e4 may be substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, or optionally substituted C 2~30 alkynyl, or an oxygen protecting group.

[0157] In some embodiments of any one of the above aspects, -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), SP(O)(OR e4 )2, -SP(S)(OR e4 )2, and -SP(S)(SR e5 )(OR e4) in which at least one R e4 is H and at least one R e4 is other than H.

[0158] In some embodiments of any one of the above aspects, -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(O)(OR e4 )2, -SP(S)(OR e4 )2, -SP(S)(SR e5 )(OR e4 ), and -SP(S)(SR e5 )2, all R e4 is H.

[0159] In some embodiments of any one of the above aspects, -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(O)(OR e4 )2, -SP(S)(OR e4 )2, -SP(S)(SR e5 )(OR e4 ), and -SP(S)(SR e5 )2, all R e4 is other than H.

[0160] In some embodiments of any one of the above aspects, -P(S)(SR e5 )(OR e4 ), -P(S)(SRe5 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(S)(SR e5 )(OR e4 ), and -SP(S)(SR e5 ) At least one R in 2 e5 is H.

[0161] In some embodiments of any one of the above aspects, -P(S)(SR e5 )(OR e4 ), -P(S)(SR e5 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(S)(SR e5 )(OR e4 ), and -SP(S)(SR e5 ) At least one R in 2 e5 is not H. For example, -P(S)(SR e5 )(OR e4 ), -P(S)(SR e5 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(S)(SR e5 )(OR e4 ), and -SP(S)(SR e5 ) At least one R in 2 e5 may be substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, or optionally substituted C 2~30 alkynyl, or sulfur protecting groups.

[0162] In some embodiments of any one of the above aspects, -P(S)(SR e5 )2, -OP(S)(SR e5 )2 and -SP(S)(SRe5 )2, at least one R e5 is H and at least one R e5 is other than H.

[0163] In some embodiments, -P(S)(SR e5 )(OR e4 ), -P(S)(SR e5 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(S)(SR e5 )(OR e4 ), and -SP(S)(SR e5 )2, all R e5 is H.

[0164] In some embodiments, -P(S)(SR e5 )(OR e4 ), -P(S)(SR e5 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(S)(SR e5 )(OR e4 ), and -SP(S)(SR e5 )2, all R e5 is other than H.

[0165] In some embodiments of any one of the aspects described herein, R e is optionally substituted -C 2~6 Alkenyl-R e1 For example, R e Ha-C 2~6 Alkenyl-R e1 where C 2~6Alkenyl includes OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C 8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p and R e1 is -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -P(S)(SR e5 )2, -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(O)(OR e4 )2, -SP(S)(OR e4 )2, -SP(S)(SR e5 )(OR e4 ), or -SP(S)(SR e5 )2.

[0166] In some embodiments of the various aspects described herein, R e is -CH(Rd1 )-R e1 or -C(R d1 )=CHR e1 where R d1 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted -C 2~30 alkenyl, or optionally substituted -C 2~30 alkynyl, and R e1 is -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -P(S)(SR e5 )2, -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(O)(OR e4 )2, -SP(S)(OR e4 )2, -SP(S)(SR e5 )(OR e4 ), -SP(S)(SR e5 )2, -OR e2 or -SR e3 is.

[0167] In some embodiments of any one of the aspects described herein, R e -C(R d1 )=CHR e1 -C(R d1 )=CHR e1 Note that the double bond in can be in the cis or trans configuration. Thus, in some embodiments of any one of the above aspects, R e -C(R d1 )=CHR e1 wherein the double bond is in a cis configuration. In some other embodiments of any one of the above aspects, R e -C(R d1 )=CHR e1 where the double bond is in the trans configuration.

[0168] In some embodiments of any one of the above aspects, R e is -CH=CHR e1 For example, R e is -CH=CHR e1 where the double bond is in the trans configuration. In some other examples, R e is -CH=CHR e1 where the double bond is in the cis configuration.

[0169] In some embodiments of any one of the above aspects, R e is -CH=CH-P(O)(OR e4 )2, -CH=CH-P(S)(OR e4 )2, -CH=CH-P(S)(SR e5 )(OR e4 ), -CH=CH-P(S)(SR e5 )2, -CH=CH-OP(O)(OR e4 )2, -CH=CH-OP(S)(OR e4 )2, -CH=CH-OP(S)(SR e5 )(OR e4 ), -CH=CH-OP(S)(SR e5 )2, -CH=CH-SP(O)(OR e4 )2, -CH=CH-SP(S)(OR e4 )2, -CH=CH-SP(S)(SR e5 )(OR e4 ), or -CH=CH-SP(S)(SR e5 )2. For example, R e -CH=CH-P(O)(OR e4 )2.

[0170] In some embodiments of any one of the above aspects, R e2 is a hydrogen or oxygen protecting group. For example, R e2 is hydrogen or 4,4'-dimethoxytrityl (DMT). In some preferred embodiments, R e2 is H.

[0171] In some embodiments of any one of the aspects described herein, R e is optionally substituted -C 1~6 Alkenyl-R e1 For example, R e Ha-C 1~6 Alkenyl-R e1 where C 1~6 Alkenyl includes OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C 8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p and R e1 -OR e2 , -SR e3 , -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -P(S)(SR e5 )2, -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(O)(ORe4 )2, -SP(S)(OR e4 )2, -SP(S)(SR e5 )(OR e4 ), or -SP(S)(SR e5 )2.

[0172] In some embodiments of any one of the aspects described herein, R e -CH(R e6 )-R e1 where R e1 -OR e2 , -SR e3 , -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -P(S)(SR e5 )2, -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(O)(OR e4 )2, -SP(S)(OR e4 )2, -SP(S)(SR e5 )(OR e4 ), or -SP(S)(SR e5 )2 and R e6 is H, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, or optionally substituted C 2~30 It is alkynyl.

[0173] In some embodiments of any one of the aspects described herein, R e6is H or is OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1 ~C8 alkoxy), O(C1-C8) haloalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C1-C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 30 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. In one non-limiting example, R e6 is H. In some other non-limiting examples, R e6 is a C1-C alkyl group optionally substituted with a substituent selected from NH2, OH, C(O)NH2, COOH, halo, SH, and C1-C6 alkoxy. 30 It is alkyl.

[0174] In some embodiments of any one of the aspects described herein, R e is -CH(R e6 )-OR e7 , where R e7 H, -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -P(S)(SR e5 )2, -OP(O)(OR e4)2. For example, R e -CH(R e6 )-OR e7 where R e6 is H or optionally substituted C1-C 30 alkyl, and R e7 is H or -P(O)(OR e4 )2.

[0175] In some embodiments of any one of the aspects described herein, R e -CH2-OR e2 where R e2 is a hydrogen or oxygen protecting group.

[0176] In some embodiments of any one of the aspects described herein, R e -CH(R e6 )-SR e8 where R e8 H, -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -P(S)(SR e5 )2, -OP(O)(OR e4 )2.

[0177] In some embodiments of any one of the aspects described herein, R a' is a halogen and R b is optionally substituted C 1~30 alkyl, and R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R 4 is H and R e -C(R d1 )=CHR e1 For example, R a' is F and R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl; R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R4 is H and R e -C(R d1 )=CHR e1 In some embodiments of any one of the aspects described herein, R a' is F and R b is methyl and R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R 4 is H and R e -C(R d1 )=CHR e1 is.

[0178] In some embodiments of any one of the aspects described herein, R a' is a halogen and R b is optionally substituted C 1~30 alkyl, and R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R 4 is H and R e -C(R d1 )=CHR e1 where R e1 is -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -P(S)(SR e5 )2, -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(O)(OR e4 )2, -SP(S)(OR e4 )2, -SP(S)(SR e5 )(OR e4 ), -SP(S)(SR e5 )2, -OR e2 or -SR e3 For example, R a' is F and R bis methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl; R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R 4 is H and R e -C(R d1 )=CHR e1 where R e1 is -P(O)(OR e4 )2, -P(S)(OR e4 )2, -P(S)(SR e5 )(OR e4 ), -P(S)(SR e5 )2, -OP(O)(OR e4 )2, -OP(S)(OR e4 )2, -OP(S)(SR e5 )(OR e4 ), -OP(S)(SR e5 )2, -SP(O)(OR e4 )2, -SP(S)(OR e4 )2, -SP(S)(SR e5 )(OR e4 ), or -SP(S)(SR e5 In some embodiments of any one of the aspects described herein, R a' is F and R b is methyl and R c is the bond to the internucleoside linkage to the subsequent nucleoside, and R 4 is H and R e -C(R d1 )=CHR e1 where R e1 -P(O)(OR e4 )2.

[0179] R a In some embodiments of any one of the aspects described herein, R a is halogen, hydrogen, -OR c2 , -SR c3 , optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 Alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH2CH2O) r CH2CH2OR c4 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH2CH2NH) s CH2CH2-R c5 , NHC(O)R c4 , a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, a solid support, a linker covalently attached to a solid support, or a reactive phosphorus group.

[0180] In some embodiments of any one of the above aspects, R a' -OR a2 If R a2 can be hydrogen or a hydroxyl protecting group. For example, R a2 In some embodiments of any one of the aspects described herein, can be hydrogen.

[0181] R a Ga-SR a3 If R a3 can be hydrogen or a sulfur protecting group. Thus, in some embodiments of any one of the above aspects, R a3 is hydrogen.

[0182] R a -O(CH2CH2O) m CH2CH2OR a4 where m is 1 to 50, and R a4 are independently generated for each occurrence, H, C1 to C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R a5 and Ra5 is independently at each occurrence amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0183] R a -NH(CH2CH2NH) n CH2CH2-R a5 where n is 1 to 50, and R a5 is independently at each occurrence amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0184] In some embodiments of any one of the aspects described herein, R a is hydrogen, halogen, -OR a2 or optionally substituted C1 to C 30 Alkoxy. For example, R a is halogen, -OR a2 or optionally substituted C1 to C 30 In some embodiments of any one of the aspects described herein, R a is F, OH or optionally substituted C1-C 30 It is an alkoxy.

[0185] In some embodiments of any one of the aspects described herein, R a is a halogen. For example, R a is fluoro (F).

[0186] In some embodiments of any one of the aspects described herein, R aare OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkyl). koxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C1-C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 30 alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R a is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, 30 In some embodiments of any one of the aspects described herein, R a is -O(CH2) p CH3, where p is 1 to 21. For example, p is 14, 15, 16, 17, or 18. In one non-limiting example, p is 16.

[0187] In some embodiments of any one of the above aspects, R a is -O(CH2) q R a7 where q is between 2 and 10, and R a7 is C1-C6 alkoxy, amino (NH2), CO2H, OH, or halo. For example, R a7is —CH or —NH. Thus, in some embodiments of any one of the above aspects, R a is -O(CH2) q -OMe or R a is -O(CH2) q -NH2.

[0188] In some embodiments of any one of the aspects described herein, q is 2, 3, 4, 5, or 6. For example, q is 2, 3, or 6. In one non-limiting example, q is 2. In another non-limiting example, q is 3 or 6.

[0189] In some embodiments of any one of the aspects described herein, R a is C1-C6 haloalkyl. For example, R a is C1-C4 haloalkyl. In some embodiments of any one of the aspects described herein, R a is -CF3, -CF2CF3, -CF2CF2CF3 or -CF2(CF3)2.

[0190] In some embodiments of any one of the aspects described herein, R a -OCH(CH2OR a8 )CH2OR a9 where R a8 and R a9 are independently H, optionally substituted C1 to C 30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R a8 and R a9 are independently optionally substituted C1 to C 30 It is alkyl.

[0191] In some embodiments of any one of the aspects described herein, R a -CH2C(O)NHR a10 where R a10 is H, optionally substituted C1 to C30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R a10 is H or optionally substituted C1 to C 30 In some embodiments, R a10 is an optionally substituted C1-C6 alkyl.

[0192] In some embodiments of any one of the above aspects, R a is a phosphorus(III) group. For example, R a is a reactive phosphorus(III) group.

[0193] Without wishing to be bound by theory, reactive phosphorus groups are useful for forming internucleoside linkages, including, for example, phosphodiester and phosphorothioate internucleoside linkages. Such reactive phosphorus groups are known in the art and include, for example, phosphoramidites, H-phosphonates, phosphate triesters, and phosphorus-containing chiral auxiliary groups. III or P V Contains phosphorus atoms in the valence state. Phosphoramidites (P III Reactive phosphorus(III) groups in the form of (III)-( ...

[0194] In some embodiments of any one of the aspects described herein, the reactive phosphate group is —OP(OR P )N(R P2 )2, -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SRP )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 For example, a reactive phosphorus(III) group is -OP(OR P )N(R P2 )2.

[0195] In some embodiments of any one of the above aspects, R P is optionally substituted C 1~6 alkyl. For example, R P may be substituted with C 1~6 It is alkyl.

[0196] OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkyl) oxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p1, 2, 3, 4, or 5 substituents independently selected from —NH or CH-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5, or 6. In some embodiments, R p may be substituted by CN or -SC(O)Ph, C 1~6 alkyl. For example, R p is cyanoethyl (-CH2CH2CN).

[0197] In the reactive phosphorus(III) group, each R P2 are independently optionally substituted C 1~6 For example, each R P2 can be independently selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl. P2 Note that when groups are present, they can be the same or different. Thus, in some non-limiting examples, two or more R P2 If groups are present, then those R P2 In some other non-limiting examples, two or more R P2 If groups are present, then those R P2 In some embodiments of any one of the above aspects, each R P2 is isopropyl.

[0198] In some embodiments of any one of the above aspects, both R P2taken together with the nitrogen atom to which they are attached, form an optionally substituted 3- to 8-membered heterocyclyl. Exemplary heterocyclyls include, but are not limited to, pyrrolidinyl, piperazinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolizinyl, 1,4-diazaperhydroepynyl, 1,3-dioxanyl, 1,4-dioxanyl, and the like, each of which is optionally substituted with OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, silyl, and the like. Ano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p It may be substituted with 1, 2 or 3 substituents independently selected from -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0199] In some embodiments of any one of the above aspects, R P and R P2and one of the following, taken together with the atom(s) to which they are attached, form an optionally substituted 4- to 8-membered heterocyclyl. Exemplary heterocyclyls include, but are not limited to, pyrrolidinyl, piperazinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolidinyl, 1,4-diazaperhydroepynyl, 1,3-dioxanyl, 1,4-dioxanyl, and the like, each of which is optionally substituted with OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C (1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p It may be substituted with 1, 2 or 3 substituents independently selected from -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0200] In the reactive phosphorus(III) group, each R P3 are independently optionally substituted C 1~6 alkyl. For example, R P3are OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkyl). koxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 1~6 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R P3 is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl, each of which is optionally substituted with NH, OH, C(O)NH, COOH, halo, SH, or C-C alkoxy.

[0201] In some embodiments of any one of the above aspects, the reactive phosphorus(III) group is —OP(OR P )N(R P2 )2. For example, a reactive phosphorus(III) group is -OP(OR P )N(R P2 )2, where R P is cyanoethyl (-CH2CH2CN), and each R P2 is isopropyl.

[0202] In some embodiments of any one of the aspects described herein, R a is a reactive phosphorus group. For example, R a -OP(OR P )N(R P2 )2, -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 is.

[0203] In some embodiments of any one of the above aspects, R 3 -OP(OR P )N(R P2 )2, -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 where R P is optionally substituted C 1~6alkyl, and each R P2 are independently optionally substituted C 1~6 alkyl, and each R P3 are independently optionally substituted C 1~6 It is alkyl.

[0204] In some embodiments of any one of the above aspects, R a -OP(OR P )N(R P2 )2. For example, R a -OP(OR P )N(R P2 )2, where R P is cyanoethyl (-CH2CH2CN), and each R P2 is isopropyl.

[0205] In some embodiments of any one of the aspects described herein, R a can be a linker to a solid support.

[0206] In some embodiments of any one of the aspects described herein, R a is a halogen and R b is optionally substituted C 1~30 alkyl. For example, R a is F and R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl. a is F and R b is methyl.

[0207] R 3 In some embodiments of any one of the aspects described herein, R 3 is hydrogen, halogen, -OR c2 , -SR c3 , optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 Alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH2CH2O) r CH2CH2OR c4 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH2CH2NH) s CH2CH2-R c5 , NHC(O)R c4 , a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, a solid support, a linker covalently attached to a solid support, or a reactive phosphorus group.

[0208] In some embodiments of any one of the aspects described herein, R 3 is a reactive phosphorus group. For example, R 3 -OP(OR P )N(R P2 )2, -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 is.

[0209] In some embodiments of any one of the above aspects, R 3-OP(OR P )N(R P2 )2, -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P ) optionally substituted C 1~6 alkyl, and each R P2 are independently optionally substituted C 1~6 alkyl, and each R P3 are independently optionally substituted C 1~6 It is alkyl.

[0210] In some embodiments of any one of the above aspects, R 3 -OP(OR P )N(R P2 )2. For example, R 3 -OP(OR P )N(R P2 )2, where R P is cyanoethyl (-CH2CH2CN), and each R P2 is isopropyl.

[0211] In some embodiments of any one of the aspects described herein, R 3 is a solid support or a linker covalently attached to a solid support.

[0212] In some embodiments of any one of the above aspects, R 3 -OR c2 If R c2 can be hydrogen or a hydroxyl protecting group. For example, R c2 In some embodiments of any one of the aspects described herein, can be hydrogen.

[0213] R3 Ga-SR c3 If R c3 can be hydrogen or a sulfur protecting group. Thus, in some embodiments of any one of the above aspects, R c3 is hydrogen.

[0214] R 3 -O(CH2CH2O) r CH2CH2OR c4 where r can be 1 to 50, and R c4 are independently generated for each occurrence, H, C1 to C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R c5 and R c5 is independently at each occurrence amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0215] R 3 But -NH(CH2CH2NH) s CH2CH2-R c5 where s can be 1 to 50, and R c5 can be, independently at each occurrence, amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0216] In some embodiments of any one of the aspects described herein, R 3 is hydrogen, halogen, -OR c2 or optionally substituted C1 to C 30 Alkoxy. For example, R 3 is halogen, -OR c2 or optionally substituted C1 to C 30 In some embodiments of any one of the aspects described herein, R 3 is F, OH or optionally substituted C1-C 30 It is an alkoxy.

[0217] In some embodiments of any one of the aspects described herein, R 3 are OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkyl). koxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C1-C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 30 alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R 3 is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, 30 In some embodiments of any one of the aspects described herein, R 3 is -O(CH2) t CH3, where t is 1 to 21. For example, t is 14, 15, 16, 17, or 18. In one non-limiting example, t is 16.

[0218] In some embodiments of any one of the above aspects, R 3 is -O(CH2) u R c7 where u is 2 to 10, and R a7is C1-C6 alkoxy, amino (NH2), CO2H, OH, or halo. For example, R c7 is —CH or NH. Thus, in some embodiments of any one of the aspects described herein, R 3 is -O(CH2) u -OMe or R c is -O(CH2) u It is NH2.

[0219] In some embodiments of any one of the aspects described herein, u is 2, 3, 4, 5, or 6. For example, u is 2, 3, or 6. In one non-limiting example, u is 2. In another non-limiting example, u is 3 or 6.

[0220] In some embodiments of any one of the aspects described herein, R 3 is C1-C6 haloalkyl. For example, R 3 is C1-C4 haloalkyl. In some embodiments of any one of the aspects described herein, R 3 is -CF3, -CF2CF3, -CF2CF2CF3 or -CF2(CF3)2.

[0221] In some embodiments of any one of the aspects described herein, R 3 -OCH(CH2OR c8 )CH2OR c8 where R c8 and R c9 are independently H, optionally substituted C1 to C 30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R c8 and R c9 are independently optionally substituted C1 to C 30 It is alkyl.

[0222] In some embodiments of any one of the aspects described herein, R c-CH2C(O)NHR c10 where R c10 is H, optionally substituted C1 to C 30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R c10 is H or optionally substituted C1 to C 30 In some embodiments, R a10 is an optionally substituted C1-C6 alkyl.

[0223] In some embodiments of any one of the aspects described herein, R a is a halogen and R b is optionally substituted C 1~30 alkyl, and R 3 is a hydroxyl, a protected hydroxyl, a reactive phosphorus group (e.g., -OP(OR P )N(R P2 )2, -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 ), a solid support, or a linker covalently linked to a solid support. For example, R a' is F and R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl; R 3is a hydroxyl, a protected hydroxyl, a reactive phosphorus group (e.g., -OP(OR P )N(R P2 )2, -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 ), a solid support, or a linker covalently linked to a solid support. a' is F and R b is methyl and R 3 is a hydroxyl, a protected hydroxyl, a reactive phosphorus group (e.g., -OP(OR P )N(R P2 )2, -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3), a solid support, or a linker covalently linked to a solid support.

[0224] In some embodiments of any one of the aspects described herein, R a is a halogen and R b is optionally substituted C 1~30 alkyl, and R 3 represents a hydroxyl, a protected hydroxyl, -OP(OR P )N(R P2 ) 2, or a linker covalently linked to a solid support. For example, R a' is F and R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, and R 3 represents a hydroxyl, a protected hydroxyl, -OP(OR P )N(R P2 )2, or a linker covalently linked to a solid support. In some embodiments of any one of the aspects described herein, R a' is F and R b is methyl and R 3 represents a hydroxyl, a protected hydroxyl, -OP(OR P )N(R P2 )2, or a linker covalently linked to a solid support.

[0225] R 5 In some embodiments of the various aspects described herein, R 5 may be substituted C 1~6 Alkyl-R 5a , optionally substituted -C 2~6 Alkenyl-R 5a , or optionally substituted -C 2~6 Alkynyl-R 5a where R 5a -OR 5b , -SR 5c , hydrogen, a phosphorus(III) group, a solid support, or a linker to a solid support.5a -OR 5b If R 5b can be H or a hydroxyl protecting group. Similarly, R 5a Ga-SR 5c If R 5c can be H or a sulfur protecting group.

[0226] In some embodiments of any one of the aspects described herein, R 5 -CH(R 5d )-R 5a where R 5d is hydrogen, halogen, or optionally substituted C1-C 30 Alkyl, optionally substituted C2-C 30 Alkenyl, optionally substituted C2-C 30 Alkynyl or optionally substituted C1-C 30 It is an alkoxy.

[0227] In some embodiments of any one of the above aspects, R 5 -CH(R 5d )-R 5a If R 5d is H or is OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1 ~C8 alkoxy), O(C1-C8) haloalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2)p C1-C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 30 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R 5d is H. In some other non-limiting examples, R 5d is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, 30 It is alkyl.

[0228] In some embodiments of the various aspects described herein, R 5 -CH(R 5d )-OR 5b where R 5d is H or is OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1 ~C8 alkoxy), O(C1-C8) haloalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C1-C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 30 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R 5dis H. In some other non-limiting examples, R 5d is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, 30 It is alkyl.

[0229] In some embodiments of the various aspects described herein, R 5 may be substituted C 1~6 Alkyl-R 5a , or optionally substituted -C 2~6 Alkenyl-R 5a is.

[0230] In some embodiments of any one of the aspects described herein, R 5 -C(R 5d )=CHR 5a -C(R 5d )=CHR 5a Note that the double bond in can be in the cis or trans configuration. Thus, in some embodiments of any one of the above aspects, R d -C(R 5d )=CHR 5a wherein the double bond is in a cis configuration. In some other embodiments of any one of the above aspects, R d -C(R 5d )=CHR 5a where the double bond is in the trans configuration.

[0231] In some embodiments of any one of the aspects described herein, R 5 is -CH=CHR 5a For example, R 5 is -CH=CHR 5a where the double bond is in the trans configuration. In some other non-limiting examples, R 5 is -CH=CHR 5a where the double bond is in the cis configuration.

[0232] In some embodiments of any one of the above aspects, R5 -C(R 5d )=CHR 5a If R 5d is H or is OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1 ~C8 alkoxy), O(C1-C8) haloalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C1-C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 30 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6; R 5a is a phosphorus(III) group. For example, R 5 is -CH=CHR 5a is.

[0233] In some embodiments of any one of the aspects described herein, R 5a is a reactive phosphorus(III) group.

[0234] In some embodiments of any one of the above aspects, R 5a is -P(O)(OR 5e )2, -P(S)(OR 5e )2, -P(S)(SR 5f )(OR 5e ), -P(S)(SR 5f)2, -OP(O)(OR 5e )2, -OP(S)(OR 5e )2, -OP(S)(SR 5f )(OR 5e ), -OP(S)(SR 5f )2, -SP(O)(OR 5e )2, -SP(S)(OR 5e )2, -SP(S)(SR 5f )(OR 5e ), or -SP(S)(SR 5f )2, where each R 5e are independently hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 alkynyl, an oxygen protecting group, or an alkali metal or transition metal group with a total charge of +1, and each R 5f are independently hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, or optionally substituted C 2~30 alkynyl, or sulfur protecting groups.

[0235] In some embodiments of any one of the above aspects, R 5 is -CH=CH-P(O)(OR 5e )2, -CH=CH-P(S)(OR 5e )2, -CH=CH-P(S)(SR 5f )(OR 5e ), -CH=CH-P(S)(SR 5f )2, -CH=CH-OP(O)(OR 5e )2, -CH=CH-OP(S)(OR 5e )2, -CH=CH-OP(S)(SR 5f )(OR 5e ), -CH=CH-OP(S)(SR 5f )2, -CH=CH-SP(O)(OR 5e )2, -CH=CH-SP(S)(OR 5e )2, -CH=CH-SP(S)(SR 5f )(OR 5e), or -CH=CH-SP(S)(SR 5f )2, where each R 5e are independently hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 alkynyl, an oxygen protecting group, or an alkali metal or transition metal group with a total charge of +1, and each R 5f are independently hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, or optionally substituted C 2~30 alkynyl, or sulfur protecting groups.

[0236] In some embodiments of any one of the above aspects, -P(O)(OR 5e )2, -P(S)(OR 5e )2, -P(S)(SR 5f )(OR 5e ), -OP(O)(OR 5e )2, -OP(S)(OR 5e )2, -OP(S)(SR 5f )(OR 5e ), SP(O)(OR 5e )2, -SP(S)(OR 5e )2, and -SP(S)(SR 5f )(OR 5e ) with at least one R 5e is hydrogen.

[0237] In some other embodiments of any one of the above aspects, -P(O)(OR 5e )2, -P(S)(OR 5e )2, -P(S)(SR 5f )(OR 5e ), -OP(O)(OR 5e )2, -OP(S)(OR 5e )2, -OP(S)(SR 5f )(OR 5e ), SP(O)(OR 5e )2, -SP(S)(OR 5e )2, or -SP(S)(SR5f )(OR 5e ) with at least one R 5e is not hydrogen. For example, P(O)(OR 5e )2, -P(S)(OR 5e )2, -P(S)(SR 5f )(OR 5e ), -OP(O)(OR 5e )2, -OP(S)(OR 5e )2, -OP(S)(SR 5f )(OR 5e ), SP(O)(OR 5e )2, -SP(S)(OR 5e )2, and -SP(S)(SR 5f )(OR 5e ) at least one R 5e may be substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, or optionally substituted C 2~30 alkynyl, or an oxygen protecting group.

[0238] In some embodiments of any one of the above aspects, -P(O)(OR 5e )2, -P(S)(OR 5e )2, -P(S)(SR 5f )(OR 5e ), -OP(O)(OR 5e )2, -OP(S)(OR 5e )2, -OP(S)(SR 5f )(OR 5e ), SP(O)(OR 5e )2, -SP(S)(OR 5e )2, and -SP(S)(SR 5f )(OR 5e ) in which at least one R 5e is H and at least one R 5e is other than H.

[0239] In some embodiments of any one of the above aspects, -P(O)(OR 5e )2, -P(S)(OR 5e )2, -P(S)(SR 5f)(OR 5e ), -OP(O)(OR 5e )2, -OP(S)(OR 5e )2, -OP(S)(SR 5f )(OR 5e ), -OP(S)(SR 5f )2, -SP(O)(OR 5e )2, -SP(S)(OR 5e )2, -SP(S)(SR 5f )(OR 5e ), and -SP(S)(SR 5f )2, all R 5e is H.

[0240] In some embodiments of any one of the above aspects, -P(O)(OR 5e )2, -P(S)(OR 5e )2, -P(S)(SR 5f )(OR 5e ), -OP(O)(OR 5e )2, -OP(S)(OR 5e )2, -OP(S)(SR 5f )(OR 5e ), -OP(S)(SR 5f )2, -SP(O)(OR 5e )2, -SP(S)(OR 5e )2, -SP(S)(SR 5f )(OR 5e ), and -SP(S)(SR 5f )2, all R 5e is other than H.

[0241] In some embodiments of any one of the above aspects, -P(S)(SR 5f )(OR 5e ), -P(S)(SR 5f )2, -OP(S)(OR 5e )2, -OP(S)(SR 5f )(OR 5e ), -OP(S)(SR 5f )2, -SP(S)(SR 5f )(OR 5e ), and -SP(S)(SR 5f ) At least one R in 25f is H.

[0242] In some embodiments of any one of the above aspects, -P(S)(SR 5f )(OR 5e ), -P(S)(SR 5f )2, -OP(S)(OR 5e )2, -OP(S)(SR 5f )(OR 5e ), -OP(S)(SR 5f )2, -SP(S)(SR 5f )(OR 5e ), and -SP(S)(SR 5f ) At least one R in 2 5f is other than H. For example, -P(S)(SR 5f )(OR 5e ), -P(S)(SR 5f )2, -OP(S)(OR 5e )2, -OP(S)(SR 5f )(OR 5e ), -OP(S)(SR 5f )2, -SP(S)(SR 5f )(OR 5e ), and -SP(S)(SR 5f ) At least one R in 2 5f may be substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, or optionally substituted C 2~30 alkynyl, or sulfur protecting groups.

[0243] In some embodiments of any one of the above aspects, -P(S)(SR 5f )2, -OP(S)(SR 5f )2, and -SP(S)(SR 5f )2, at least one R 5f is H and at least one R 5f is other than H.

[0244] In some embodiments, -P(S)(SR 5f )(OR 5e ), -P(S)(SR5f )2, -OP(S)(OR 5e )2, -OP(S)(SR 5f )(OR 5e ), -OP(S)(SR 5f )2, -SP(S)(SR 5f )(OR 5e ), and -SP(S)(SR 5f )2, all R 5f is H.

[0245] In some embodiments, -P(S)(SR 5f )(OR 5e ), -P(S)(SR 5f )2, -OP(S)(OR 5e )2, -OP(S)(SR 5f )(OR 5e ), -OP(S)(SR 5f )2, -SP(S)(SR 5f )(OR 5e ), and -SP(S)(SR 5f )2, all R 5f is other than H.

[0246] In some embodiments of any one of the above aspects, R 5 -CH=CH-P(O)(OR 5e )2, where each R 5e is H or an oxygen protecting group.

[0247] In some embodiments of any one of the above aspects, R 5 -CH=CH-P(O)(OR 5e )2, where the double bond is in a trans configuration. In some other embodiments of any one of the above aspects, R 5 -CH=CH-P(O)(OR 5e )2, where the double bond is in the cis configuration.

[0248] In some embodiments of any one of the aspects described herein, R a is a halogen and R b is optionally substituted C 1~30alkyl, and R 3 is a hydroxyl, a protected hydroxyl, a reactive phosphorus group (e.g., -OP(OR P )N(R P2 )2, -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 ), a solid support, or a linker covalently linked to a solid support; R 4 is H and R 5 -CH=CH-P(O)(OR 5e )2. For example, R a' is F and R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl; R 3 is a hydroxyl, a protected hydroxyl, a reactive phosphorus group (e.g., -OP(OR P )N(R P2 )2, -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P)R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 ), a solid support, or a linker covalently linked to a solid support; R 4 is H and R 5 -CH=CH-P(O)(OR 5e In some embodiments of any one of the aspects described herein, R a' is F and R b is methyl and R 3 is a hydroxyl, a protected hydroxyl, a reactive phosphorus group (e.g., -OP(OR P )N(R P2 )2, -OP(SR P )N(R P2 )2, -OP(O)(OR P )N(R P2 )2, -OP(S)(OR P )N(R P2 )2, -OP(O)(SR P )N(R P2 )2, -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 ), a solid support, or a linker covalently linked to a solid support; R 4 is H and R 5 -CH=CH-P(O)(OR 5e )2.

[0249] In some embodiments of any one of the aspects described herein, R a is a halogen and R b is optionally substituted C 1~30 alkyl, and R 3 represents a hydroxyl, a protected hydroxyl, -OP(ORP )N(R P2 ) 2, or a linker covalently linked to a solid support, R 4 is H and R 5 -CH=CH-P(O)(OR 5e )2. For example, R a' is F and R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl; R 3 represents a hydroxyl, a protected hydroxyl, -OP(OR P )N(R P2 ) 2, or a linker covalently linked to a solid support, R 4 is H and R 5 -CH=CH-P(O)(OR 5e In some embodiments of any one of the aspects described herein, R a' is F and R b is methyl and R 3 represents a hydroxyl, a protected hydroxyl, -OP(OR P )N(R P2 ) 2, or a linker covalently linked to a solid support, R 4 is H and R 5 -CH=CH-P(O)(OR 5e )2.

[0250] Internucleoside linkage As used herein, "internucleoside linkage" refers to a covalent linkage between adjacent nucleosides. Two major classes of internucleoside linkages are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing linkages include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-), siloxane (-O-Si(H)2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified internucleoside linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotide compounds compared to natural phosphodiester linkages. In certain embodiments, linkages having chiral atoms can be prepared as racemic mixtures, as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus(III)-containing and non-phosphorus(III)-containing linkages are well known to those skilled in the art.

[0251] The phosphate group in the internucleoside linkage can be modified by replacing one of its oxygen atoms with a different substituent. One result of this modification can be increased resistance of the oligonucleotide to nucleolytic breakdown. Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphodiester internucleoside linkage can be replaced by any of the following: S, Se, BR3 (R is hydrogen, alkyl, aryl), C (i.e., alkyl group, aryl group, etc.), H, NR2 (R is hydrogen, optionally substituted alkyl, aryl), or OR (R is optionally substituted alkyl or aryl). The phosphorus(III) atom in the unmodified phosphate group is achiral. However, if one of the non-bridging oxygens is replaced with one of the atoms or groups of atoms described above, the phosphorus(III) atom becomes chiral. In other words, the phosphorus(III) atom in the phosphate group modified in this manner is an asymmetric center. The asymmetric phosphorus(III) atom can have either the "R" configuration (referred to herein as Rp) or the "S" configuration (referred to herein as Sp).

[0252] In phosphorodithioates, both non-bridging oxygens are replaced with sulfur. The phosphorus center in phosphorodithioates is achiral, which eliminates the formation of oligonucleotide diastereomers. Therefore, without wishing to be bound by theory, modification of both non-bridging oxygens to remove chiral centers, such as forming phosphorodithioates, may be desirable in that it cannot produce diastereomeric mixtures. The non-bridging oxygens can be independently O, S, Se, B, C, H, N, or OR (R is alkyl or aryl).

[0253] Phosphodiester internucleoside linkages can also be modified by replacement of the bridging oxygen (i.e., the oxygen linking the phosphate to the sugar of the nucleoside) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylenesulfonates). Replacement can be at one of the linking oxygens or at both linking oxygens. When the bridging oxygen is the 3'-oxygen of the nucleoside, replacement with carbon is preferred. When the bridging oxygen is the 5'-oxygen of the nucleoside, replacement with nitrogen is preferred.

[0254] Modified phosphate linkages in which at least one of the oxygens linked to the phosphate is replaced or the phosphate group is replaced with a non-phosphorus(III) group are also referred to as "non-phosphodiester intersugar linkages" or "non-phosphodiester linkers."

[0255] In certain embodiments, the phosphate group can be replaced with a non-phosphorus-containing connector, e.g., a dephospho linker. A dephospho linker is also referred to herein as a non-phosphodiester linker. Without wishing to be bound by theory, because the charged phosphodiester group is the reactive center in nucleolytic degradation, its replacement with a neutral structural mimic should confer enhanced nuclease stability. Again, without wishing to be bound by theory, in some embodiments it may be desirable to introduce a modification in which the charged phosphate group is replaced with a neutral moiety.

[0256] Examples of moieties that can replace the phosphate group include amide (e.g., amide-3 (3'-CH2-C(=O)-N(H)-5') and amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylic acid ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonic acid ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3'-O-CH2-O-5'), oxime, methyleneimino, methykenecarbonylamino, amino), methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether (C3'-O-C5'), thioether (C3'-S-C5'), thioacetamide (C3'-N(H)-C(=O)-CH2-S-C5', C3'-OP(O)-O-SS-C5', C3'-CH2-NH-NH-C5', 3'-NHP(O)(OCH3)-O-5' and 3'-NHP(O)(OCH3)-O-5', as well as non-ionic linkages containing mixed N, O, S and CH2 component parts. For example, "Carbohydrate See "Modifications in Antisense Research," edited by YS Sanghvi and PD Cook, ACS Symposium Series 580, Chapters 3 and 4 (pp. 40-65). Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amide, carbamate, and ethylene oxide linkers.

[0257] It is well known to those skilled in the art that in certain instances, replacement of a non-bridging oxygen can lead to enhanced cleavage of the intersugar linkage by the adjacent 2'-OH, and therefore modification of the non-bridging oxygen may in many instances require modification of the 2'-OH, e.g., a modification that does not participate in cleavage of the adjacent intersugar linkage, e.g., arabinose sugars, 2'-O-alkyl, 2'-F, LNA, and ENA.

[0258] Preferred non-phosphodiester internucleoside linkages include phosphorothioates, phosphorothioates in an enantiomeric excess of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95% or more for the Sp isomer, phosphorothioates in an enantiomeric excess of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95% or more for the Rp isomer, phosphorodithioates, phosphotriesters, aminoalkylphosphotrioesters, alkylphosphonates (e.g., methylphosphonates), selenophosphates, phosphoramidates (e.g., N-alkylphosphoramidates), and boranophosphonates.

[0259] Further exemplary non-phosphorus-containing internucleoside linking groups are described in U.S. Pat. Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, Nos. 5,596,086, 5,602,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, 5,792,608, 5,646,269 and 5,677,439, the contents of each of which are incorporated herein by reference.

[0260] In some embodiments of any one of the above aspects, the oligonucleotides of the invention comprise one or more neutral internucleoside linkages that are non-ionic. Suitable neutral internucleoside linkages include phosphotriester, methylphosphonate, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), and thioformacetal (3'-S-CH2-O-5'); non-ionic linkages containing siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters, and / or amides (see, e.g., "Carbohydrate Modifications in Antisense Research," edited by Y.S. Sanghvi and P.D. Cook, ACS Symposium Series, Vol. 1, No. 1, pp. 111-114, 2002). 580, Chapters 3 and 4 (pp. 40-65)); and non-ionic linkers containing mixed N, O, S, and CH2 component parts.

[0261] In one embodiment, the non-phosphodiester backbone linkages are selected from the group consisting of phosphorothioate, phosphorodithioate, alkyl-phosphonate, and phosphoramidate backbone linkages.

[0262] In some embodiments of any one of the aspects described herein, the internucleoside linkage is: TIFF2024539093000017.tif25128, where R IL1 and R IL2 is, independently at each occurrence, absent, O, S, CH, NR (where R is hydrogen, alkyl, aryl), or optionally substituted alkylene, where the alkylene backbone can contain one or more of O, S, SS, and NR (where R is hydrogen, alkyl, aryl) internally and / or terminally; R IL3 and R IL4are each independently O, OR (R is hydrogen, alkyl, or aryl), S, Se, BR3 (R is hydrogen, alkyl, or aryl), BH3 - , C (i.e., alkyl, aryl, etc.), H, NR2 (where R is hydrogen, alkyl, aryl), alkyl, or aryl. R IL1 and R IL2 replaces the oxygen linked to the 5' carbon of the first nucleoside sugar, and R IL1 and R IL2 of course, the other of these replaces the oxygen linked to the 3' (or 2') carbon of the second nucleoside sugar.

[0263] In some embodiments of any one of the above aspects, R IL1 , R IL2 , R IL1 and R IL2 are all O.

[0264] In some embodiments, R IL1 and R IL2 is O and R IL3 and R IL4 For example, R IL3 and R IL4 one of which is S and the other is O, or R IL3 and R IL4 Both are S.

[0265] In some embodiments of any one of the above aspects, R a' or R c One of them is R IL1 or R IL2 For example, R c is R IL1 is a bond to

[0266] In some embodiments of any one of the above aspects, R a' or R c One of them is R IL1 or R IL2 is a bond to Rd is R IL1 or R IL2 For example, R c is R IL1 is a bond to R d is R IL2 is a bond to

[0267] Nitrogen Protecting Groups Some embodiments of the various aspects described herein include a nitrogen protecting group (also referred to herein as an amino protecting group). Nitrogen protecting groups include -OH, -OR NP1 , -N(R NP2 )2, -C(=O)R NP1 , -C(=O)N(R NP2 )2, -CO2R NP1 , -SO2R NP1 , -C(=NR NP2 )R NP1 , -C(=NR NP2 ) OR NP1 , -C(=NR NP2 )N(R NP2 )2, -SO2N(R NP2 )2, -SO2R NP2 , -SO2OR NP2 , -SOR NP1 , -C(=S)N(R NP2 )2, -C(=O)SR NP2 , -C(=S)SR NP2 , C 1~10 Alkyl (e.g., aralkyl, heteroaralkyl), C 2~10 Alkenyl, C 2~10 Alkynyl, C 3~10 Carbocyclyl, 3- to 14-membered heterocyclyl, C 6~14 aryl, and 5- to 14-membered heteroaryl groups, where each R NP1 independently, C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, Hetero C 1~10 Alkyl, Hetero C 2~10 Alkenyl, Hetero C 2~10 Alkynyl, C 3~10Carbocyclyl, 3- to 14-membered heterocyclyl, C 6~14 aryl, or 5- to 14-membered heteroaryl, or two R NP1 groups taken together form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, and each R NP2 are independently hydrogen, C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, Hetero C 1~10 Alkyl, Hetero C 2~10 Alkenyl, Hetero C 2~10 Alkynyl, C 3~10 Carbocyclyl, 3- to 14-membered heterocyclyl, C 6~14 aryl, and 5- to 14-membered heteroaryl, or two R SP3 groups taken together form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, where R NP1 and R NP2 Each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl may optionally be OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8 ) alkyl, O(C1-C8) alkyl (i.e., C1-C8 alkoxy), O(C1-C8) haloalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) pIt may be substituted with 1, 2, 3, 4 or 5 substituents independently selected from -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0268] Nitrogen protecting groups are well known in the art and include those detailed in "Greene's Protecting Groups in Organic Synthesis," PGM Wuts, 5th Edition, John Wiley & Sons, 2014, incorporated herein by reference.

[0269] Exemplary amides (e.g., —C(═O)R NP1 ) Nitrogen protecting groups include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

[0270] Exemplary carbamates (e.g., —C(═O)OR NP1) Nitrogen protecting groups include methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2- (2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate mate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4-dimethylthiophenylcarbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropylcarbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chloro Monylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzylthiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2, 2-Dimethoxyacylvinylcarbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propylcarbamate, 1,1-dimethylpropynylcarbamate, di(2-pyridyl)methylcarbamate, 2-furanylmethylcarbamate, 2-iodoethylcarbamate, isobornylcarbamate, isobutylcarbamate, isonicotinylcarbamate, p-(p'-methoxyphenylazo)benzylcarbamate, 1-methylcyclobutylcarbamate, 1 -methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,Examples include, but are not limited to, 6-trimethylbenzylcarbamate.

[0271] Exemplary sulfonamides (e.g., —S(═O)R NP1 ) Nitrogen protecting groups include, for example, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6 -dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0272] Further exemplary nitrogen protecting groups include phenothiazinyl-(10)-acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuNP2inimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1 ,1,4,4-Tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo- 3-Pyrrolin-3-yl)amine, quaternary ammonium salt, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1- Dimethylthiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,These include, but are not limited to, 5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane and N-diphenylborinic acid derivatives, N-[phenyl(pentNP1cylchromium- or tungsten)acyl]amine, N-copper chelates, N-zinc chelates, N-nitroamines, N-nitrosamines, amine N-oxides, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkylphosphoramidates, dibenzylphosphoramidate, diphenylphosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridine sulfenamide (Npys).

[0273] Oxygen Protecting Groups Some embodiments of the various aspects described herein include an oxygen protecting group (also referred to herein as a hydroxyl protecting group). Oxygen protecting groups include -R OP1 , -N(R OP2 )2, -C(=O)SR OP1 , -C(=O)R OP1 , -CO2R OP1 , -C(=O)N(R OP2 )2, -C(=NR OP2 )R OP1 , -C(=NR OP2 ) OR OP1 , -C(=NR OP2 )N(R OP2 )2, -S(=O)R OP1 , -SO + 2nd Round OP1 , -Si(R OP1 )3, -P(R OP3 )2, -P(R OP3 ) + 3X - , -P(OR OP3 )2, -P(OR OP3 )3X - , -P(=O)(R OP1)2, -P(=O)(OR OP3 )2, and -P(=O)(N(R OP2 )2)2, where each X - is a counterion, and each R OP1 independently, C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, Hetero C 1~10 Alkyl, Hetero C 2~10 Alkenyl, Hetero C 2~10 Alkynyl, C 3~10 Carbocyclyl, 3- to 14-membered heterocyclyl, C 6~14 aryl, or 5- to 14-membered heteroaryl, or two R OP1 groups taken together form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, and each R OP2 is hydrogen, -OH, -OR OP1 , -N(R OP3 )2, -CN, -C(=O)R OP1 , -C(=O)N(R OP3 )2, -CO2R OP1 , -SO2R OP1 , -C(=NR OP3 ) OR OP1 , -C(=NR OP3 )N(R OP3 )2, -SO2N(R OP3 )2, -SO2R OP3 , -SO2OR OP3 , -SOR OP1 , -C(=S)N(R OP3 )2, -C(=O)SR OP3 , -C(=S)SR OP3 , -P(=O)(R OP1 )2, -P(=O)(OR OP3 )2, -P(=O)(N(R OP3 )2)2, C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, Hetero C 1~10 Alkyl, Hetero C 2~10 Alkenyl, Hetero C2~10 Alkynyl, C 3~10 Carbocyclyl, 3- to 14-membered heterocyclyl, C 6~14 aryl, and 5- to 14-membered heteroaryl, or two R OP2 groups taken together form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, and each R OP3 are independently hydrogen, C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, Hetero C 1~10 Alkyl, Hetero C 2~10 Alkenyl, Hetero C 2~10 Alkynyl, C 3~10 Carbocyclyl, 3- to 14-membered heterocyclyl, C 6~14 aryl, and 5- to 14-membered heteroaryl, or two R OP3 groups taken together form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, where R OP1 , R OP2 and R OP3 Each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl may optionally be OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8 ) alkyl, O(C1-C8) alkyl (i.e., C1-C8 alkoxy), O(C1-C8) haloalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2)p It may be substituted with 1, 2, 3, 4 or 5 substituents independently selected from -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0274] Oxygen protecting groups are well known in the art and include those detailed in "Greene's Protecting Groups in Organic Synthesis," PGM Wuts, 5th Edition, John Wiley & Sons, 2014, incorporated herein by reference.

[0275] Exemplary oxygen protecting groups include methyl, t-butyloxycarbonyl (BOC or Boc), methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-Methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4- methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trimethyl- Lithoethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-Dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4''-tris(levulinoyloxyphenyl)methyl, 4,4',4''-tris(benzoyloxyphenyl)methyl nyl)methyl, 3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), di Ethyl isopropyl silyl (DEIPS), dimethylthexyl silyl, t-butyl dimethyl silyl (TBDMS), t-butyl diphenyl silyl (TBDPS), tribenzyl silyl, tri-p-xylyl silyl, triphenyl silyl, diphenyl methyl silyl (DPMS), t-butyl methoxyphenyl silyl (TBMPS), formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenyl methoxyacetate, pheno oxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyl dithioacetal), adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-Trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate Carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthio) (Methoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monos(SP3inoate), (E)-2-methyl Examples of suitable alkyl esters include, but are not limited to, methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).

[0276] In some embodiments of any one of the aspects described herein, the oxygen protecting group is benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthin-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In certain embodiments, T1 is a hydroxyl protecting group selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and dimethoxytrityl, with the more preferred hydroxyl protecting group T1 being 4,4'-dimethoxytrityl.

[0277] Sulfur Protecting Groups Some embodiments of the various aspects described herein include a sulfur protecting group (also referred to herein as a thiol protecting group). The sulfur protecting group includes -R SP1 , -N(R SP2 )2, -C(=O)SR SP1 , -C(=O)R SP1 , -CO2R SP1 , -C(=O)N(R SP2 )2, -C(=NR SP2 )R SP1 , -C(=NR SP2 ) OR SP1 , -C(=NR SP2 )N(R SP2 )2, -S(=O)R SP1 , -SO2R SP1 , -Si(R SP1 )3, -P(R SP3 )2, -P(R SP3 ) + 3X - , -P(OR SP3 )2, -P(OR SP3 ) + 3X - , -P(=O)(R SP1 )2, -P(=O)(OR SP3 )2, and -P(=O)(N(R SP2 )2)2, including, but not limited to, X - is a counterion, and each R SP1independently, C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, Hetero C 1~10 Alkyl, Hetero C 2~10 Alkenyl, Hetero C 2~10 Alkynyl, C 3~10 Carbocyclyl, 3- to 14-membered heterocyclyl, C 6~14 aryl, or 5- to 14-membered heteroaryl, or two R SP1 groups taken together form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, and each R SP2 is hydrogen, -OH, -OR SP1 , -N(R SP3 )2, -CN, -C(=O)R SP1 , -C(=O)N(R SP3 )2, -CO2R SP1 , -SO2R SP1 , -C(=NR SP3 ) OR SP1 , -C(=NR SP3 )N(R SP3 )2, -SO2N(R SP3 )2, -SO2R SP3 , -SO2OR SP3 , -SOR SP1 , -C(=S)N(R SP3 )2, -C(=O)SR SP3 , -C(=S)SR SP3 , -P(=O)(R SP1 )2, -P(=O)(OR SP3 )2, -P(=O)(N(R SP3 )2)2, C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, Hetero C 1~10 Alkyl, Hetero C 2~10 Alkenyl, Hetero C 2~10 Alkynyl, C 3~10 Carbocyclyl, 3- to 14-membered heterocyclyl, C 6~14 aryl, and 5- to 14-membered heteroaryl, or two R SP2groups taken together form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, and each R SP3 are independently hydrogen, C 1~10 Alkyl, C 1~10 Perhaloalkyl, C 2~10 Alkenyl, C 2~10 Alkynyl, Hetero C 1~10 Alkyl, Hetero C 2~10 Alkenyl, Hetero C 2~10 Alkynyl, C 3~10 Carbocyclyl, 3- to 14-membered heterocyclyl, C 6~14 aryl, and 5- to 14-membered heteroaryl, or two R SP3 groups taken together form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, where R SP1 , R SP2 and R SP3 The alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl groups are optionally substituted with OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8 ) alkyl, O(C1-C8) alkyl (i.e., C1-C8 alkoxy), O(C1-C8) haloalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p It may be substituted with 1, 2, 3, 4 or 5 substituents independently selected from -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0278] Sulfur protecting groups are well known in the art and include those detailed in "Greene's Protecting Groups in Organic Synthesis," PGM Wuts, 5th Edition, John Wiley & Sons, 2014, incorporated herein by reference.

[0279] Compounds / Monomers In one aspect, provided herein are compounds / monomers, i.e., 2'-geminal substituted nucleosides and nucleotides of formula (III) or (III'). In some embodiments, the compound of formula (III) is selected from the compounds shown in Table 1.

[0280] Table 1: Exemplary compounds of formula (III) TIFF2024539093000018.tif248153

[0281] In some embodiments, the compound of formula (III) is selected from the compounds shown in Tables 2-3.

[0282] Table 2: Exemplary compounds of formula (III) TIFF2024539093000019.tif196170TIFF2024539093000020.tif112170

[0283] Table 3: Exemplary compounds of formula (III) TIFF2024539093000021.tif134128TIFF2024539093000022.tif134128

[0284] In the compounds of Table 3, one or both hydroxyls attached to phosphorus(III) are independently replaced by optionally substituted C1-C 30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30For example, one of the hydroxyl groups attached to phosphorus(III) can be replaced with an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl. 60 It can be substituted with alkynyl.

[0285] Exemplary 2'-geminal substituted nucleosides and nucleotides of formula (III) can be prepared according to the synthetic schemes shown in Figures 1-15.

[0286] In some embodiments of any one of the above aspects, the compound of Formula (III) is compound 10, 11, 11A, 12, 13, or 13A shown in FIG.

[0287] In some embodiments of any one of the above aspects, the compound of Formula (III) is compound 32 or 33 shown in FIG.

[0288] In some embodiments of any one of the above aspects, the compound of Formula (III) is compound 37 or 38 shown in FIG.

[0289] In some embodiments of any one of the above aspects, the compound of Formula (III) is compound 42 or 43 shown in FIG.

[0290] In some embodiments of any one of the above aspects, the compound of Formula (III) is compound 55, 56, or 56A shown in FIG.

[0291] In some embodiments of any one of the above aspects, the compound of Formula (III) is compound 60, 61A, 61B, 62A, 62B, 63A, or 63B shown in FIG.

[0292] In some embodiments of any one of the above aspects, the compound of Formula (III) is compound 64A, 64B, 64A, or 65B shown in FIG.

[0293] In some embodiments of any one of the above aspects, the compound of Formula (III) is compound 69, 70A, 70B, 71A, 71B, 72A, or 72B shown in FIG.

[0294] In some embodiments of any one of the above aspects, the compound of Formula (III) is compound 73A, 73B, 74A, or 74B shown in FIG.

[0295] In some embodiments of any one of the above aspects, the compound of Formula (III) is compound 15, 16, 17, 18, 19, 20, or 21 shown in FIG.

[0296] In some embodiments of any one of the above aspects, the compound of Formula (III) is compound 22, 23, 24, 25, 26, or 27 shown in FIG.

[0297] In some embodiments of any one of the above aspects, the compound of Formula (III) is compound 75, 76, 77, 78, 79, 80, or 81 shown in FIG.

[0298] In some embodiments of any one of the above aspects, the compound of Formula (III) is compound 82, 83, 84, 85, 86, or 87 shown in FIG.

[0299] In some embodiments of any one of the above aspects, the compound of Formula (III) is not: R a is F and R b is methyl and R 3 -OR c2 , a reactive phosphorus(III) group or a linker to a solid support, and R c2 is hydrogen or a hydroxyl protecting group, and R 4 is H and R 5 Ga-CH2OR 5b and R 5b is H, a hydroxyl protecting group or a phosphorus group, and B is adenine, cytosine, guanine or uracil, each of which can be unprotected, protected or modified.

[0300] In some embodiments of any one of the above aspects, the compound of Formula (III) is not: R a is OH and R b is methyl, vinyl or ethynyl, and R 3 -OR c2 , a reactive phosphorus(III) group or a linker to a solid support, and R c2 is hydrogen or a hydroxyl protecting group, and R 4 is H and R 5 Ga-CH2OR 5b and R 5b is H, a hydroxyl protecting group or a phosphorus group, and B is adenine or guanine, which can each be unprotected, protected or modified.

[0301] In some embodiments of any one of the above aspects, the compound of formula (III) is not compounds 1 to 9, 14, 28 to 31, 34 to 36, 39 to 41, 44 to 55, 57 to 59, or 66 to 68 shown in FIGS.

[0302] In some embodiments of any one of the aspects described herein, the compound of Formula (III) has the structure Not the compound in TIFF2024539093000023.tif24128, where: R b is hydrogen or substituted or unsubstituted C1-C4 alkyl, R c -OR Ix where: R Ix is H, -P(O)(OM)2, -P(O)(OM)-OP(O)(OM)2, -P(O)(Oalkyl)2, -P(O)(Oalkyl)-OP(O)(Oalkyl)2, -PO3H2, -PO3HM, -PO3M2, -PO2SH2, -PO2SHM, -PO2SM2, -PO3M, or -PO2SM, protecting group, ligand, ligand-bearing monomer, -F, -(C rC6) alkyl, -(C2-C6) aryl, -(C(R 3 )2) n OR 3 , -(C(R 3 )2) n SR 3 , -(C(R 3 )2) n N(R 3 )2, -(C(R 3 )2) n C(O)N(R 3 )2, -(C(R 3 )2) n O(C r C6) alkyl, -(C(R 3 )2) n S(C r C6) alkyl, -(C(R 3 )2) n O(C(R 3 )2) n N((Ci~C6) alkyl)2, -(C(R 3 )2) n ON((Ci~C6) alkyl)2, -C(O)R 3 , -C(O)R 3 C(O)H, -C(O)R 3 C(O)OH, -C(O)R 3 C(O)R 3 , -C(O)R 3 C(O)NR 3 -PO2, -P(OR 3 )2, -P(N(R 3 )2)2, -P(OR 3 )N(R 3 )2, or a linker, R 4 is H, R 5x is H, -P(O)(OM)2, -P(O)(OM)-OP(O)(OM)2, -P(O)(Oalkyl)2, -P(O)(Oalkyl)-OP(O)(Oalkyl)2, -PO3H2, -PO3HM, -PO3M2, -PO2SH2, -PO2SHM, -PO2SM2, -PO3M, or -PO2SM, a protecting group, a ligand, or a ligand-bearing monomer; M, independently at each occurrence, represents an alkali or transition metal with a total charge of +1; and n is an integer of 1 to 4.

[0303] In some embodiments of any one of the aspects described herein, the compound of formula (III) Not TIFF2024539093000024.tif37163.

[0304] Oligonucleotides In one aspect, provided herein is an oligonucleotide comprising (i) at least one 2'-geminal substituted nucleoside of formula (I) or (I'), and / or (ii) a 2'-geminal substituted nucleoside of formula (II) or (II') at the 5'-terminal nucleotide.

[0305] In some embodiments of any one of the aspects described herein, the 2′-geminal substituted nucleoside of Formula (II) at the 5′-terminal nucleotide has the structure: TIFF2024539093000025.tif21128 nucleosides, where: R b is hydrogen or substituted or unsubstituted C1-C4 alkyl, R c -OR is the bond to the internucleotide linkage to the subsequent nucleoside Ix where: R Ix is H, -P(O)(OM)2, -P(O)(OM)-OP(O)(OM)2, -P(O)(Oalkyl)2, -P(O)(Oalkyl)-OP(O)(Oalkyl)2, -PO3H2, -PO3HM, -PO3M2, -PO2SH2, -PO2SHM, -PO2SM2, -PO3M, or -PO2SM, protecting group, ligand, ligand-bearing monomer, -F, -(C r C6) alkyl, -(C2-C6) aryl, -(C(R 3 )2) n OR 3 , -(C(R 3 )2)n SR 3 , -(C(R 3 )2) n N(R 3 )2, -(C(R 3 )2) n C(O)N(R 3 )2, -(C(R 3 )2) n O(C r C6) alkyl, -(C(R 3 )2) n S(C r C6) alkyl, -(C(R 3 )2) n O(C(R 3 )2) n N((Ci~C6) alkyl)2, -(C(R 3 )2) n ON((Ci~C6) alkyl)2, -C(O)R 3 , -C(O)R 3 C(O)H, -C(O)R 3 C(O)OH, -C(O)R 3 C(O)R 3 , -C(O)R 3 C(O)NR 3 -PO2, -P(OR 3 )2, -P(N(R 3 )2)2, -P(OR 3 )N(R 3 )2, or a linker, R 4 is H, R e is -CH2OR IIx where: R IIx is -H, -P(O)(OM)2, -P(O)(OM)-OP(O)(OM)2, -P(O)(Oalkyl)2, -P(O)(Oalkyl)-OP(O)(Oalkyl)2, -PO3H2, -PO3HM, -PO3M2, -PO2SH2, -PO2SHM, -PO2SM2, -PO3M, or -PO2SM, a protecting group, a ligand, or a ligand-bearing monomer; M, independently at each occurrence, represents an alkali or transition metal with a total charge of +1; and n is an integer of 1 to 4.

[0306] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II) is Not TIFF2024539093000026.tif31167.

[0307] In some embodiments of any one of the aspects described herein, the 2′-geminal substituted nucleoside of Formula (I) has the structure: TIFF2024539093000027.tif24128 nucleosides, where: R b is hydrogen or substituted or unsubstituted C1-C4 alkyl, R c -OR is the bond to the internucleotide linkage to the subsequent nucleoside Ix where: R Ix is H, -P(O)(OM)2, -P(O)(OM)-OP(O)(OM)2, -P(O)(Oalkyl)2, -P(O)(Oalkyl)-OP(O)(Oalkyl)2, -PO3H2, -PO3HM, -PO3M2, -PO2SH2, -PO2SHM, -PO2SM2, -PO3M, or -PO2SM, protecting group, ligand, ligand-bearing monomer, -F, -(C r C6) alkyl, -(C2-C6) aryl, -(C(R 3 )2) n OR 3 , -(C(R 3 )2) n SR 3 , -(C(R 3 )2) n N(R 3 )2, -(C(R 3 )2) n C(O)N(R 3 )2, -(C(R 3 )2) n O(C r C6) alkyl, -(C(R 3 )2) n S(C r C6) alkyl, -(C(R 3)2) n O(C(R 3 )2) n N((Ci~C6) alkyl)2, -(C(R 3 )2) n ON((Ci~C6) alkyl)2, -C(O)R 3 , -C(O)R 3 C(O)H, -C(O)R 3 C(O)OH, -C(O)R 3 C(O)R 3 , -C(O)R 3 C(O)NR 3 -PO2, -P(OR 3 )2, -P(N(R 3 )2)2, -P(OR 3 )N(R 3 )2, or a linker, R 4 is H, R d is the bond to the internucleotide linkage to the preceding nucleoside, M, independently at each occurrence, represents an alkali or transition metal with a total charge of +1; and n is an integer from 1 to 4, and 1, and n is an integer of 1 to 4.

[0308] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (I) is Not TIFF2024539093000028.tif31128.

[0309] It should be noted that the 2'-geminal substituted nucleoside of Formula (I) or (I') can be located anywhere in the oligonucleotide. In some embodiments, the 2'-geminal substituted nucleoside of Formula (I) or (I') is located at positions 2-10, counting from the 5' end of the oligonucleotide. For example, the 2'-geminal substituted nucleoside of Formula (I) or (I') is located at position 2, or position 3, position 4, position 5, position 6, position 7, position 8, position 9, or position 10, counting from the 5' end of the oligonucleotide. In some non-limiting examples, the 2'-geminal substituted nucleoside of Formula (I) or (I') is located at position 2, counting from the 5' end of the oligonucleotide. In some non-limiting examples, the 2'-geminal substituted nucleoside of Formula (I) or (I') is located at position 3, counting from the 5' end of the oligonucleotide. In some non-limiting examples, the 2'-geminal substituted nucleoside of Formula (I) or (I') is at position 4, counting from the 5' end of the oligonucleotide. In some non-limiting examples, the 2'-geminal substituted nucleoside of Formula (I) or (I') is at position 5, counting from the 5' end of the oligonucleotide. In some non-limiting examples, the 2'-geminal substituted nucleoside of Formula (I) or (I') is at position 6, counting from the 5' end of the oligonucleotide. In some non-limiting examples, the 2'-geminal substituted nucleoside of Formula (I) or (I') is at position 7, counting from the 5' end of the oligonucleotide. In some non-limiting examples, the 2'-geminal substituted nucleoside of Formula (I) or (I') is at position 8, counting from the 5' end of the oligonucleotide. In some non-limiting examples, the 2'-geminal substituted nucleoside of Formula (I) or (I') is at position 9, counting from the 5' end of the oligonucleotide. In some non-limiting examples, the 2'-geminal substituted nucleoside of Formula (I) or (I') is at position 10, counting from the 5' end of the oligonucleotide.

[0310] In some embodiments, an oligonucleotide comprises at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-geminal substituted nucleosides of formula (I) and / or (I'). For example, an oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-geminal substituted nucleosides of formula (I) and / or (I'). In some embodiments, all nucleotides in the oligonucleotide are 2'-geminal substituted nucleosides described herein. In other words, an oligonucleotide comprises only 2'-geminal substituted nucleotides of formula (I), (I'), (II) and / or (II').

[0311] In some embodiments of any one of the aspects described herein, the oligonucleotide includes only 2'-geminally substituted nucleotides of Formulas (I) and (II).

[0312] In some embodiments of any one of the aspects described herein, the oligonucleotide comprises only 2'-geminally substituted nucleotides of Formulas (I) and (II), and the oligonucleotide further comprises a ligand, e.g., monovalent or polyvalent N-acetylgalactosamine (GalNac), linked to the oligonucleotide. For example, the oligonucleotide comprises only 2'-geminally substituted nucleotides of Formulas (I) and (II), and the oligonucleotide further comprises a ligand, e.g., monovalent or polyvalent N-acetylgalactosamine (GalNac), linked to its 3' end.

[0313] In some embodiments, the 5' terminal nucleotide of the oligonucleotide is a 2'-geminal substituted nucleotide of formula (II) or (II'). In some other embodiments, the 5' terminal nucleotide of the oligonucleotide is not a 2'-geminal substituted nucleotide of formula (II) or (II').

[0314] In some embodiments, the oligonucleotide further comprises a nucleoside having a modified sugar. By "modified sugar" is meant a sugar or moiety other than a 2'-deoxy (i.e., 2'-H), 2'-OH ribose sugar, or a 2'-geminal substituted nucleoside described herein. Exemplary nucleotides containing modified sugars include 2'-F ribose, 2'-OMe ribose, 2'-O,4'-C-methylene ribose (locked nucleic acid, LNA), anhydrohexitol (1,5-anhydrohexitol nucleic acid, HNA), cyclohexene (cyclohexene nucleic acid, CeNA), 2'-methoxyethyl ribose, 2'-O-allyl ribose, 2'-C-allyl ribose, 2'-ON-methylacetamido (2'-O-NMA) ribose, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) ribose, 2'-O-aminopropyl (2'-O-AP) ribose, 2'-F arabinose (2'-ara-F), threose (threose nucleic acid, TNA), and 2,3-dihydroxypropyl (glycol nucleic acid, GNA). It should be noted that the nucleoside having a modified sugar can be present at any position in the oligonucleotide.

[0315] In some embodiments of any one of the aspects described herein, the oligonucleotide, e.g., the antisense strand or sense strand of the dsRNA described herein, comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, CeNA nucleotides or analogs thereof. In some embodiments, the CeNA nucleotides or analogs thereof TIFF2024539093000029.tif66170, where: R is F, Cl, Br, I, H, protected OH, OMe, F, O-MOE, O-alkyl, O-alkene, O-alkyne, OC 16 , a branched lipid, or a protected aminoalkyl; R 1 are F, Cl, Br, I, H, protected OH, OMe, F, O-MOE, O-alkyl, O-alkene, O-alkyne, OC 16, a branched lipid, a protected aminoalkyl, and B is a nucleobase.

[0316] In some embodiments of any one of the aspects described herein, the antisense or sense strand of the dsRNA described herein comprising an oligonucleotide, e.g., a CeNA nucleotide or analog thereof, TIFF2024539093000030.tif141159, where: R' is H or CH3; R is F, Cl, Br, I, H, protected OH, OMe, F, O-MOE, O-alkyl, O-alkene, O-alkyne, OC 16 , a branched lipid, or a protected aminoalkyl; R 1 are F, Cl, Br, I, H, protected OH, OMe, F, O-MOE, O-alkyl, O-alkene, O-alkyne, OC 16 , a branched lipid, a protected aminoalkyl, PG is a protecting group, and B is a nucleobase.

[0317] Some exemplary CeNA nucleosides and nucleotides and their analogs are described in Kumar et al. Nucleic Acids Research, 2020, 48, 4028-4040; Declercq et al. J. Am. Chem. Soc. 2002, 124, 928-933; Egli et al., J. Am. Chem. Soc. 2011, 133, 16642-16649; Wang et al., J. Am. Chem. Soc. 2000, 122, 8595-8602; Wan et al., J. Med. Chem. 2016, 59, 9645-9667; Ermolinsky et al., Russian Journal of Bioorganic Chemistry, 2002, 28, 50-57; Beheraet al., J. Am. Chem. Soc. 2020, 142, 456-467; Ghotekar et al., Org. Lett. 2020, 22, 537-541; Deshpande et al., Tetrahedron Letters 2004, 45, 2255-2258; Deshpande et al., Tetrahedron 2007, 63, 602-608; Deshpande et al., Carbohydrate Research 2008, 343, 1163-1170; Sanki et al., Tetrahedron 2008, 64, 10406-10416; and Rao et al., J. Org. Chem. 2015, 80, 1499-1505, the contents of all of which are incorporated herein by reference in their entireties.

[0318] In some embodiments, the oligonucleotide further comprises at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 2'-fluoro (2'-F) nucleotide. For example, the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-F nucleotides. Note that the 2'-F nucleotide can be present at any position in the oligonucleotide.

[0319] In some embodiments, the oligonucleotide comprises 2'-geminal substituted nucleosides and 2'-F nucleosides, eg, comprises only 2'-geminal substituted nucleosides and 2'-F nucleosides.

[0320] In some embodiments, the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, 2'-OMe nucleotide. For example, the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-OMe nucleotides. Note that the 2'-OMe nucleotide can be present at any position in the oligonucleotide.

[0321] In some embodiments, the oligonucleotide comprises 2'-geminal substituted nucleosides and 2'-OMe nucleosides, e.g., comprises only 2'-geminal substituted nucleosides and 2'-OMe nucleosides. In some other embodiments, the oligonucleotide comprises 2'-geminal substituted nucleosides, 2'-OMe nucleosides and 2'-F nucleosides, e.g., comprises only 2'-geminal substituted nucleosides, 2'-OMe nucleosides and 2'-F nucleosides.

[0322] In some embodiments, the oligonucleotide further comprises at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-deoxy, e.g., 2'-H nucleotides. For example, the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-deoxy, e.g., 2'-H nucleotides. Note that the 2'-deoxy, e.g., 2'-H nucleotides can be present at any position in the oligonucleotide. For example, the oligonucleotide can comprise 2'-deoxy, e.g., 2'-H nucleotides at 1, 2, 3, 4, 5, or 6 of positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the oligonucleotide. In some embodiments, the oligonucleotide comprises 2'-deoxy nucleotides at positions 5 and 7, counting from the 5' end of the oligonucleotide.

[0323] In some embodiments, the oligonucleotide comprises 2'-geminally substituted nucleosides and 2'-deoxy (2'-H) nucleotides, e.g., comprises only 2'-geminally substituted nucleosides and 2'-deoxy (2'-H) nucleotides. In some embodiments, the oligonucleotide comprises 2'-geminally substituted nucleosides, 2'-OMe nucleosides, and 2'-deoxy (2'-H) nucleotides, e.g., comprises only 2'-geminally substituted nucleosides, 2'-OMe nucleosides, and 2'-deoxy (2'-H) nucleotides. In some embodiments, the oligonucleotide comprises 2'-geminally substituted nucleosides, 2'-F nucleosides, and 2'-deoxy (2'-H) nucleotides, e.g., comprises only 2'-geminally substituted nucleosides, 2'-F nucleosides, and 2'-deoxy (2'-H) nucleotides. In some embodiments, the oligonucleotide comprises 2'-geminal substituted nucleosides, 2'-OMe nucleosides, 2'-F nucleosides and 2'-deoxy (2'-H) nucleotides, e.g., comprises only 2'-geminal substituted nucleosides, 2'-OMe nucleosides, 2'-F nucleosides and 2'-deoxy (2'-H) nucleotides.

[0324] In some embodiments, an oligonucleotide can include one or more nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, that comprise a non-natural nucleobase. Nucleotides that comprise a non-natural nucleobase can be present anywhere in the oligonucleotide.

[0325] In some embodiments of any one of the above aspects, the oligonucleotide can include one or more modified internucleoside linkages, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more. For example, the oligonucleotide can include 1, 2, 3, 4, 5, or 6 modified internucleoside linkages. For example, the oligonucleotide includes 1, 2, 3, or 4 modified internucleoside linkages. In some embodiments, the oligonucleotide includes at least two modified internucleoside linkages within the first five nucleotides counting from the 5' end of the oligonucleotide and further includes at least two modified internucleoside linkages within the first five nucleotides counting from the 3' end of the oligonucleotide. For example, the oligonucleotide includes modified internucleoside linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 5' end of the oligonucleotide, and between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 3' end of the oligonucleotide.

[0326] In some embodiments of any one of the above aspects, the modified internucleoside linkage is phosphorothioate. Thus, in some embodiments of any one of the above aspects, the oligonucleotide comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more, phosphorothioate internucleoside linkages. For example, the oligonucleotide comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. For example, the oligonucleotide comprises 1, 2, 3, or 4 phosphorothioate internucleoside linkages. In some embodiments, the oligonucleotide comprises at least two phosphorothioate internucleoside linkages within the first five nucleotides counting from the 5' end of the oligonucleotide and further comprises at least two phosphorothioate internucleoside linkages within the first five nucleotides counting from the 3' end of the oligonucleotide. For example, the oligonucleotide comprises modified internucleoside linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, counting from the 5' end of the oligonucleotide, and between nucleotides 1 and 2 and between nucleotides 2 and 3, counting from the 3' end of the oligonucleotide.

[0327] In some embodiments, the oligonucleotide further comprises a ligand conjugated to the oligonucleotide.

[0328] In some embodiments, the oligonucleotide further comprises a solid support linked to the oligonucleotide.

[0329] The oligonucleotides described herein can range in length from a few nucleotides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) to hundreds of nucleotides. For example, oligonucleotides can be 5 to 100 nucleotides in length. In some embodiments, oligonucleotides are 10 to 50 nucleotides in length. For example, oligonucleotides are 15-35, more commonly 18-25, even more commonly 19-24, and most commonly 19-21 base pairs in length. In some embodiments, longer oligonucleotides, 25-30 nucleotides in length, are preferred. In some embodiments, shorter oligonucleotides, 10-15 nucleotides in length, are preferred. In another embodiment, the oligonucleotide is at least 21 nucleotides in length.

[0330] double stranded RNA Those skilled in the art are well aware that double-stranded RNAs containing 20-23 base pairs, especially 21 base pairs, are recognized to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, other researchers have found that shorter or longer double-stranded oligonucleotides can also be effective.

[0331] Therefore, in one aspect, the present invention provides a double-stranded RNA (dsRNA) comprising a first strand (also referred to as antisense strand or guide strand) and a second strand (also referred to as sense strand or passenger strand), wherein at least one of the first strand (i.e., antisense strand) or the second strand (i.e., sense strand) is the oligonucleotide described herein.In other words, at least one of the first strand (i.e., antisense strand) or the second strand (i.e., sense strand) comprises at least one 2'-geminal substituted nucleotide of formula (I) and / or (II).

[0332] In some preferred embodiments, the antisense strand is an oligonucleotide described herein. In other words, the antisense strand comprises at least one 2'-geminal substituted nucleotide of formula (I), (I'), (II) and / or (II').

[0333] In some embodiments of the various aspects described herein, the antisense strand is substantially complementary to a target nucleic acid, e.g., a target gene or a target mRNA gene, and the dsRNA is capable of directing targeted cleavage of the target nucleic acid.

[0334] Each strand of the dsRNA molecule can be in the range of 15 to 35 nucleotides in length. For example, each strand can be 17 to 35 nucleotides, 17 to 30 nucleotides, 25 to 35 nucleotides, 27 to 30 nucleotides, 17 to 23 nucleotides, 17 to 21 nucleotides, 17 to 19 nucleotides, 19 to 25 nucleotides, 19 to 23 nucleotides, 19 to 21 nucleotides, 21 to 25 nucleotides, or 21 to 23 nucleotides in length. Without limitation, the sense and antisense strands can be equal or unequal in length. For example, the sense and antisense strands can independently be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

[0335] In some embodiments, the antisense strand is 15-35 nucleotides in length. In some embodiments, the antisense strand is 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length. For example, the antisense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. In some embodiments, the antisense strand is 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. For example, the antisense strand is 21, 22, 23, 24, or 25 nucleotides in length. In some specific embodiments, the antisense strand is 22, 23, or 24 nucleotides in length, e.g., the antisense strand is 23 nucleotides in length.

[0336] Like the antisense strand, the sense strand can be 15 to 35 nucleotides in length in some embodiments. In some embodiments, the sense strand is 15 to 35, 17 to 35, 17 to 30, 25 to 35, 27 to 30, 17 to 23, 17 to 21, 17 to 19, 19 to 25, 19 to 23, 19 to 21, 21 to 25, 21 to 25, or 21 to 23 nucleotides in length. For example, the sense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. In some embodiments, the sense strand is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. For example, the sense strand is 19, 20, 21, 22, or 23 nucleotides in length. In some specific embodiments, the sense strand is 20, 21, or 22 nucleotides in length. For example, the sense strand is 21 nucleotides in length.

[0337] In some embodiments, the sense strand can be 15-35 nucleotides in length, and the antisense strand can be 15-35 nucleotides in length independently of the sense strand. In some embodiments, the sense strand is 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length, and the antisense strand is independently 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length. For example, the sense strand and the antisense strand can be independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. In some embodiments, the sense strand and the antisense strand can be independently 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. For example, the sense strand is 19, 20, 21, 22, or 23 nucleotides in length, and the antisense strand is 21, 22, 23, 24, or 25 nucleotides in length. In some specific embodiments, the sense strand is 20, 21, or 22 nucleotides in length, and the antisense strand is 22, 23, or 24 nucleotides in length. For example, the sense strand is 21 nucleotides in length, and the antisense strand is 23 nucleotides in length.

[0338] The sense strand and the antisense strand typically form a double-stranded or duplexed region. Without limitation, the duplexed region of the dsRNA agents described herein can be 12 to 35 nucleotide (or base) pairs in length. For example, the duplexed region can be 14 to 35 nucleotide pairs, 17 to 30 nucleotide pairs, 25 to 35 nucleotide pairs, 27 to 35 nucleotide pairs, 17 to 23 nucleotide pairs, 17 to 21 nucleotide pairs, 17 to 19 nucleotide pairs, 19 to 25 nucleotide pairs, 19 to 23 nucleotide pairs, 19 to 21 nucleotide pairs, 21 to 25 nucleotide pairs, or 21 to 23 nucleotide pairs in length. In another example, the duplexed region is selected from the group consisting of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length. In some embodiments, the duplex region is 18, 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs in length. For example, the duplex region is 19, 20, 21, 22 or 23 nucleotide pairs in length. In some embodiments, the duplex region is 20, 21 or 22 nucleotide pairs in length. For example, the dsRNA molecule has a duplex region of 21 base pairs.

[0339] As described herein, the dsRNA molecules described herein can comprise at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-geminal substituted nucleotides of formula (I) and / or (II).Without being limited, all of the 2'-geminal substituted nucleotides can be present in one strand.The 2'-geminal substituted nucleotides can be present at any nucleotide position in any strand, in the sense strand or antisense strand, or in both strands.

[0340] In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 2'-geminally substituted nucleotides described herein. The 2'-geminally substituted nucleotides described herein can be present at any position in the antisense strand. For example, the 2'-geminally substituted nucleotides described herein can be present in the terminal region of the antisense strand. For example, the 2'-geminally substituted nucleotides described herein can be present at one or more of positions 1, 2, 3, and 4, counting from the 5' end of the antisense strand. In another non-limiting example, the 2'-geminally substituted nucleotides described herein can be present at one or more of positions 1, 2, 3, 4, 5, and 6, counting from the 3' end of the antisense strand. In some embodiments, the 2'-geminally substituted nucleotides described herein can be present at one or more of positions 18, 19, 20, 21, 22, and 23, counting from the 5' end of the antisense strand. The 2'-geminal substituted nucleotides described herein can also be located in the central region of the antisense strand. For example, the 2'-geminal substituted nucleotides described herein can be located at one or more of positions 6, 7, 8, 9, 10, 11, 12, and 13, counting from the 5' end of the antisense strand.

[0341] In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 2'-geminally substituted nucleotides described herein. The 2'-geminally substituted nucleotides described herein can be located at any position in the sense strand. For example, the 2'-geminally substituted nucleotides described herein can be located in the terminal regions of the sense strand. For example, the 2'-geminally substituted nucleotides described herein can be located at one or more of positions 1, 2, 3, and 4, counting from the 5' end of the sense strand. In another non-limiting example, the 2'-geminally substituted nucleotides described herein can be located at one or more of positions 1, 2, 3, and 4, counting from the 3' end of the sense strand. In some embodiments, the 2'-geminally substituted nucleotides can be located at one or more of positions 18, 19, 20, and 21, counting from the 5' end of the sense strand. The 2'-geminally substituted nucleotides described herein can also be located in the central region of the sense strand. For example, a 2'-geminal substituted nucleotide described herein can be located at one or more of positions 6, 7, 8, 9, 10, 11, 12, and 13, counting from the 5' end of the sense strand. In some embodiments, the sense strand does not include a 2'-geminal substituted nucleotide.

[0342] As described herein, a dsRNA agent can include one or more nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, containing modified sugars. Thus, in some embodiments, a dsRNA agent can include one or more nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, independently selected from the group consisting of 2'-F, 2-OMe, acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), and 2'-ara-F. Nucleotides containing modified sugars can be present anywhere in a dsRNA molecule. For example, a nucleotide containing a modified sugar can be present in the sense strand, or a nucleotide containing a modified sugar can be present in the antisense strand. When more than one nucleotide containing a modified sugar is present in a dsRNA molecule, they can all be present in the sense strand, all in the antisense strand, or all in both the sense and antisense strands.

[0343] As described herein, the dsRNA molecules described herein can contain at least one 2'-fluoro (2'-F) nucleotide, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In some embodiments, the sense strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-fluoro nucleotides. The 2'-fluoro nucleotide can be located anywhere in the sense strand. For example, the sense strand contains a 2'-fluoro nucleotide at position 10, counting from the 5' end of the sense strand. In some embodiments, the sense strand contains a 2'-fluoro nucleotide at position 10, counting from the 5' end of the sense strand, and the sense strand further contains a 2'-fluoro nucleotide at position 8, 9, 11 or 12, counting from the 5' end of the sense strand. For example, the sense strand contains a 2'-fluoro nucleotide at position 9-10, counting from the 5' end of the sense strand. In another example, the sense strand comprises 2'-fluoro nucleotides at positions 10 and 11, counting from the 5' end of the sense strand. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 9, 10, and 11, counting from the 5' end of the sense strand. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 8, 9, and 10, counting from the 5' end of the sense strand. In yet some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 10, 11, and 12, counting from the 5' end of the sense strand.

[0344] In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 7, 10 and 11 from the 5' end. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, 10 and 11 from the 5' end. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12 and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises blocks of 2, 3 or 4 2'-fluoro nucleotides.

[0345] In some embodiments, the sense strand does not contain a 2'-fluoro nucleotide at a position opposite or complementary to a thermally destabilizing modification in the antisense strand of the duplex.

[0346] In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-fluoro nucleotides. The 2'-fluoro nucleotides can be located anywhere in the antisense strand. For example, the antisense strand can comprise a 2'-fluoro nucleotide at position 14, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14 and 16, counting from the 5' end of the antisense strand. In some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 14 and 16 from the 5' end. In further embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14 and 16 from the 5' end.

[0347] In some embodiments, antisense strand comprises at least one 2'-fluoro nucleotide adjacent to destabilizing modification.For example, 2'-fluoro nucleotide can be the nucleotide at the 5' or 3' end of destabilizing modification, i.e., the nucleotide at the -1 or +1 position from the position of destabilizing modification.In some embodiments, antisense strand comprises 2'-fluoro nucleotide at each of the 5' and 3' ends of destabilizing modification, i.e., at the -1 and +1 positions from the position of destabilizing modification.In some embodiments, antisense strand comprises at least two 2'-fluoro nucleotides at the 3' end of destabilizing modification, i.e., at the +1 and +2 positions from the position of destabilizing modification.

[0348] In some embodiments, both sense strand and antisense strand comprise at least one 2'-fluoro nucleotide.2'-fluoro modification can be present on any nucleotide of sense strand or antisense strand.For example, 2'-fluoro modification can be present on all nucleotides of sense strand and / or antisense strand, or each 2'-fluoro modification can be present on sense strand or antisense strand in an alternating pattern, or sense strand or antisense strand comprises both 2'-fluoro modifications in an alternating pattern.The alternating pattern of 2'-fluoro modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of 2'-fluoro modification on sense strand can be shifted relative to the alternating pattern of 2'-fluoro modification on antisense strand.

[0349] As described herein, the dsRNA molecules described herein can comprise at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-OMe nucleotides.Without being limited, all 2'-OMe nucleotides can be present in one strand.2'-OMe nucleotides can be present in any nucleotide at any position in the strand, in the sense strand or antisense strand, or in both strands.

[0350] In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-OMe nucleotides. The 2'-OMe nucleotides can be located anywhere in the sense strand. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-OMe nucleotides. The 2'-OMe nucleotides can be located anywhere in the antisense strand.

[0351] As described herein, the dsRNA molecules described herein can contain at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2'-deoxy, e.g., 2'-H ribose, nucleotides. For example, the dsRNA can contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-deoxy, e.g., 2'-H nucleotides. The 2'-deoxy nucleotides can be present at any nucleotide in any position of the strand, in the sense strand or the antisense strand, or in both strands.

[0352] As described herein, the dsRNA can include at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more, 2'-deoxy modifications in the central region of the sense and / or antisense strand. For example, at least one of the sense strand and the antisense strand can include at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more, 2'-deoxy modifications at positions 5-17, e.g., positions 6-16, 6-15, 6-14, 6-13, 6-12, 7-15, 7-14, 7-13, 7-12, 8-16, 8-15, 8-14, 8-13, 8-12, 9-16, 9-15, 9-14, 9-13, 9-12, 10-16, 10-15, 10-14, 10-13, or 10-12, counting from the 5' end of the sense or antisense strand.

[0353] In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5 or 6 2'-deoxynucleotides. For example, the antisense strand can comprise 2, 3, 4, 5 or 6 2'-deoxynucleotides. The 2'-deoxynucleotides can be located anywhere in the antisense strand. For example, the antisense strand comprises 2'-deoxynucleotides at 1, 2, 3, 4, 5 or 6 of positions 2, 5, 7, 12, 14 and 16, counting from the 5' end of the antisense strand. In a non-limiting example, the antisense strand comprises 2'-deoxynucleotides at 1, 2, 3 or 4 of positions 2, 5, 7 and 12, counting from the 5' end of the antisense strand.

[0354] In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 5 and 7, counting from the 5' end of the antisense strand. For example, the antisense strand comprises 2'-deoxynucleotides at positions 5, 7, and 12, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2, 5, and 7, counting from the 5' end of the antisense strand. For example, the antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12, and 14, counting from the 5' end of the antisense strand. For example, the antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand.

[0355] In some embodiments, the antisense comprises a 2'-deoxynucleotide at position 2 or 12, counting from the 5' end of the antisense strand. For example, the antisense comprises a 2'-deoxynucleotide at position 12, counting from the 5' end of the antisense strand.

[0356] In some embodiments, the dsRNA comprises at least three 2'-deoxy modifications, which are located at positions 2 and 14 on the antisense strand, counting from the 5' end of the antisense strand, and at position 11 on the sense strand, counting from the 5' end of the sense strand.

[0357] In some embodiments, the dsRNA comprises at least five 2'-deoxy modifications, which are located at positions 2, 12, and 14 on the antisense strand, counting from the 5' end of the antisense strand, and at positions 9 and 11 on the sense strand, counting from the 5' end of the sense strand.

[0358] In some embodiments, the dsRNA comprises at least seven 2'-deoxy modifications, which are located at positions 2, 5, 7, 12, and 14 on the antisense strand, counting from the 5' end of the antisense strand, and at positions 9 and 11 on the sense strand, counting from the 5' end of the sense strand.

[0359] In some embodiments, the antisense strand comprises at least five 2'-deoxy modifications at positions 2, 5, 7, 12, and 14, counting from the 5' end of the antisense strand.

[0360] In one non-limiting example, the sense strand does not include a 2'-deoxynucleotide at position 11, counting from the 5' end of the sense strand.

[0361] In some embodiments, the dsRNA can include one or more nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, that comprise unnatural nucleobases.

[0362] The nucleotide that comprises non-natural nucleobase can be present anywhere in dsRNA molecule.For example, the nucleotide that comprises non-natural nucleobase can be present in sense strand, or the nucleotide that comprises non-natural nucleobase can be present in antisense strand.When there are two or more nucleotides that comprise non-natural nucleobase in dsRNA molecule, they can all be present in sense strand, antisense strand, or both sense strand and antisense strand.

[0363] The dsRNA molecule described herein can further comprise at least one phosphorothioate or methylphosphonate internucleoside linkage.Phosphorothioate internucleoside linkage modification or methylphosphonate internucleoside linkage modification can be present at any position of the chain, on any nucleotide of sense strand or antisense strand or both.For example, internucleoside linkage modification can be present at every nucleotide of sense strand and / or antisense strand, or each internucleoside linkage modification can be present in an alternating pattern on sense strand or antisense strand, or sense strand or antisense strand contains both internucleoside linkage modifications in an alternating pattern.The alternating pattern of internucleoside linkage modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of internucleoside linkage modification on sense strand can be shifted relative to the alternating pattern of internucleoside linkage modification on antisense strand.

[0364] In some embodiments, dsRNA molecule comprises phosphorothioate internucleoside linkage modification or methylphosphonate internucleoside linkage modification in overhang region.For example, overhang region comprises two nucleotides with phosphorothioate internucleoside linkage or methylphosphonate internucleoside linkage between two nucleotides.Internucleoside linkage modification can be carried out to connect overhang nucleotide with the terminal paired nucleotide in double-stranded region.For example, at least 2, 3 or 4 or all overhang nucleotides can be connected with phosphorothioate internucleoside linkage or methylphosphonate internucleoside linkage, and optionally there can be an additional phosphorothioate internucleoside linkage or methylphosphonate internucleoside linkage that connects overhang nucleotide with the paired nucleotide adjacent to overhang nucleotide. For example, there may be at least two phosphorothioate internucleoside linkages between the terminal three nucleotides, two of which are overhanging nucleotides, and the third of which is a paired nucleotide adjacent to the overhanging nucleotide. Preferably, these three terminal nucleotides may be at the 3'-end of the antisense strand.

[0365] In some embodiments, the sense strand of the dsRNA molecule comprises 1 to 10 blocks of 2 to 10 phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is located at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleoside linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0366] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleoside linkages, or an antisense strand comprising either a phosphorothioate linkage, a methylphosphonate linkage, or a phosphate linkage.

[0367] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleoside linkages, or an antisense strand comprising either a phosphorothioate linkage, a methylphosphonate linkage, or a phosphate linkage.

[0368] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleoside linkages, or an antisense strand comprising either a phosphorothioate linkage, a methylphosphonate linkage, or a phosphate linkage.

[0369] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleoside linkages, or an antisense strand comprising either a phosphorothioate linkage, a methylphosphonate linkage, or a phosphate linkage.

[0370] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleoside linkages, or an antisense strand comprising either a phosphorothioate linkage, a methylphosphonate linkage, or a phosphate linkage.

[0371] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleoside linkages, or an antisense strand comprising either a phosphorothioate linkage, a methylphosphonate linkage, or a phosphate linkage.

[0372] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleoside linkages, or an antisense strand comprising either a phosphorothioate linkage, a methylphosphonate linkage, or a phosphate linkage.

[0373] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleoside linkages separated by one, two, three, or four phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleoside linkages, or an antisense strand comprising either a phosphorothioate linkage, a methylphosphonate linkage, or a phosphate linkage.

[0374] In some embodiments, the dsRNA molecules described herein further comprise one or more phosphorothioate or methylphosphonate internucleoside linkage modifications within one to ten of the terminal positions of the sense and / or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at one or both ends of the sense and / or antisense strands can be linked by phosphorothioate or methylphosphonate internucleoside linkages.

[0375] In some embodiments, the dsRNA molecules described herein comprise one or more phosphorothioate or methylphosphonate internucleoside linkage modifications within 1 to 10 internal regions of the duplex of each of the sense and / or antisense strands. For example, at positions 8 to 16 of the duplex region, counting from the 5' end of the sense strand, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked by phosphorothioate-methylphosphonate internucleoside linkages, and the dsRNA molecules can optionally further comprise one or more phosphorothioate or methylphosphonate internucleoside linkage modifications within 1 to 10 terminal positions.

[0376] In some embodiments, the dsRNA molecules described herein further comprise one to five phosphorothioate or methylphosphonate internucleoside linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and one to five phosphorothioate or methylphosphonate internucleoside linkage modifications within the last three positions, and one to five phosphorothioate or methylphosphonate internucleoside linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and one to five phosphorothioate or methylphosphonate internucleoside linkage modifications within the last six positions.

[0377] In some embodiments, the dsRNA molecules described herein further comprise one phosphorothioate internucleoside linkage modification within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate or methylphosphonate internucleoside linkage modification within the last six positions, and one phosphorothioate internucleoside linkage modification at positions 1 and 2 (counting from the 5' end) of the antisense strand and two phosphorothioate or methylphosphonate internucleoside linkage modifications within the last six positions.

[0378] In some embodiments, the dsRNA molecules described herein further comprise two phosphorothioate internucleoside linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate internucleoside linkage modification within the last six positions, and one phosphorothioate internucleoside linkage modification at positions 1 and 2 (counting from the 5' end) of the antisense strand and two phosphorothioate internucleoside linkage modifications within the last six positions.

[0379] In some embodiments, the dsRNA molecules described herein further comprise two phosphorothioate internucleoside linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleoside linkage modifications within the last four positions, and one phosphorothioate internucleoside linkage modification at positions 1 and 2 (counting from the 5' end) of the antisense strand and two phosphorothioate internucleoside linkage modifications within the last six positions.

[0380] In some embodiments, the dsRNA molecules described herein further comprise two phosphorothioate internucleoside linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleoside linkage modifications within the last four positions, and one phosphorothioate internucleoside linkage modification at positions 1 and 2 (counting from the 5' end) of the antisense strand and one phosphorothioate internucleoside linkage modification within the last six positions.

[0381] In some embodiments, the dsRNA molecules described herein further comprise one phosphorothioate internucleoside linkage modification within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate internucleoside linkage modification within the last four positions, and two phosphorothioate internucleoside linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand and two phosphorothioate internucleoside linkage modifications within the last six positions.

[0382] In some embodiments, the dsRNA molecules described herein further comprise one phosphorothioate internucleoside linkage modification within positions 1-5 (counting from the 5' end) and one within the last six positions of the sense strand, and two phosphorothioate internucleoside linkage modifications at positions 1 and 2 (counting from the 5' end) and one within the last six positions of the antisense strand.

[0383] In some embodiments, the dsRNA molecules described herein further comprise one phosphorothioate internucleoside linkage modification within positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleoside linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and one phosphorothioate internucleoside linkage modification within the last six positions.

[0384] In some embodiments, the dsRNA molecules described herein further comprise two phosphorothioate internucleoside linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and one phosphorothioate internucleoside linkage modification at positions 1 and 2 (counting from the 5' end) of the antisense strand, and two phosphorothioate internucleoside linkage modifications within the last six positions.

[0385] In some embodiments, the dsRNA molecules described herein further comprise two phosphorothioate internucleoside linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and one within the last six positions, and two phosphorothioate internucleoside linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand and one phosphorothioate internucleoside linkage modification within the last six positions.

[0386] In some embodiments, the dsRNA molecules described herein further comprise two phosphorothioate internucleoside linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate internucleoside linkage modification within the last six positions, and two phosphorothioate internucleoside linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand and two phosphorothioate internucleoside linkage modifications within the last six positions.

[0387] In some embodiments, the dsRNA molecules described herein further comprise two phosphorothioate internucleoside linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate internucleoside linkage modification within the last six positions, and one phosphorothioate internucleoside linkage modification at positions 1 and 2 (counting from the 5' end) of the antisense strand and two phosphorothioate internucleoside linkage modifications within the last six positions.

[0388] In some embodiments, the dsRNA molecules described herein further comprise two phosphorothioate internucleoside linkage modifications at positions 1 and 2 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleoside linkage modifications at positions 20 and 21, and one phosphorothioate internucleoside linkage modification at position 1 and one at position 21 (counting from the 5' end) of the antisense strand.

[0389] In some embodiments, the dsRNA molecules described herein further comprise one phosphorothioate internucleoside linkage modification at position 1 (counting from the 5' end) and one phosphorothioate internucleoside linkage modification at position 21 of the sense strand, and two phosphorothioate internucleoside linkage modifications at positions 1 and 2 and two phosphorothioate internucleoside linkage modifications at positions 20 and 21 of the antisense strand (counting from the 5' end).

[0390] In some embodiments, the dsRNA molecules described herein further comprise two phosphorothioate internucleoside linkage modifications at positions 1 and 2 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleoside linkage modifications at positions 21 and 22 of the antisense strand, and one phosphorothioate internucleoside linkage modification at position 1 and one phosphorothioate internucleoside linkage modification at position 21 of the antisense strand (counting from the 5' end).

[0391] In some embodiments, the dsRNA molecules described herein further comprise one phosphorothioate internucleoside linkage modification at position 1 (counting from the 5' end) and one phosphorothioate internucleoside linkage modification at position 21 of the sense strand, and two phosphorothioate internucleoside linkage modifications at positions 1 and 2 and two phosphorothioate internucleoside linkage modifications at positions 21 and 22 of the antisense strand (counting from the 5' end).

[0392] In some embodiments, the dsRNA molecules described herein further comprise two phosphorothioate internucleoside linkage modifications at positions 1 and 2 (counting from the 5' end) of the sense strand, two phosphorothioate internucleoside linkage modifications at positions 22 and 23, and one phosphorothioate internucleoside linkage modification at position 1 and one phosphorothioate internucleoside linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0393] In some embodiments, the dsRNA molecules described herein further comprise one phosphorothioate internucleoside linkage modification at position 1 (counting from the 5' end) and one phosphorothioate internucleoside linkage modification at position 21 of the sense strand, and two phosphorothioate internucleoside linkage modifications at positions 1 and 2 and two phosphorothioate internucleoside linkage modifications at positions 22 and 23 of the antisense strand (counting from the 5' end).

[0394] In some embodiments, the sense strand comprises at least two phosphorothioate internucleoside linkages between the first five nucleotides counting from the 5' end of the sense strand, e.g., the sense strand comprises a phosphorothioate linkage between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 5' end of the sense strand.

[0395] In some embodiments, the antisense strand comprises at least two phosphorothioate internucleoside linkages between the first five nucleotides, counting from the 5' end of the antisense strand, e.g., the antisense strand comprises a phosphorothioate linkage between nucleotides 1 and 2 and between nucleotides 2 and 3, counting from the 5' end of the antisense strand.

[0396] In some embodiments, antisense strand comprises at least two phosphorothioate internucleoside linkages between the first five nucleotides counting from the 3' end of antisense strand.For example, antisense strand comprises phosphorothioate linkages between nucleotides n and n-1 and n-2, where n is the length of antisense strand, i.e., the number of nucleotides in antisense strand.In other words, antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2 and nucleotides 2 and 3 counting from the 3' end of antisense strand.

[0397] In some embodiments, the antisense strand comprises at least two phosphorothioate internucleoside linkages within the first five nucleotides counting from the 5' end of the antisense strand, and at least two phosphorothioate internucleoside linkages within the first five nucleotides counting from the 5' end of the antisense strand, For example, the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 5' end of the antisense strand, and between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 3' end of the antisense strand.

[0398] In some embodiments, the sense strand comprises at least two phosphorothioate internucleoside linkages within the first five nucleotides counting from the 5' end of the sense strand, and the antisense strand comprises at least two phosphorothioate internucleoside linkages within the first five nucleotides counting from the 5' end of the antisense strand. For example, the sense strand comprises phosphorothioate linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 5' end of the sense strand, and the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 5' end of the antisense strand.

[0399] In some embodiments, the sense strand comprises at least two phosphorothioate internucleoside linkages within the first five nucleotides counting from the 5' end of the sense strand, and the antisense strand comprises at least two phosphorothioate internucleoside linkages within the first five nucleotides counting from the 3' end of the antisense strand. For example, the sense strand comprises phosphorothioate linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 5' end of the sense strand, and the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 3' end of the antisense strand.

[0400] In some embodiments, the dsRNA molecules described herein comprise a pattern of backbone chiral centers. In some embodiments, the common pattern of backbone chiral centers comprises at least five internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least six internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least seven internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least eight internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least nine internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least ten internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least eleven internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration.In some embodiments, the common pattern of backbone chiral centers contains eight or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains seven or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains six or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains five or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains four or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains three or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains two or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains one or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers contains eight or fewer non-chiral internucleotide linkages (phosphodiesters by way of non-limiting example). In some embodiments, the common pattern of backbone chiral centers contains seven or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers contains six or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers contains five or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers contains four or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers contains three or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers contains two or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers contains one or fewer non-chiral internucleotide linkages.In some embodiments, the common pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration and no more than 8 nonchiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration and no more than 7 nonchiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration and no more than 6 nonchiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration and no more than 6 nonchiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration and no more than 5 nonchiral internucleotide linkages. In some embodiments, the consensus pattern of backbone chiral centers includes at least 15 internucleotide linkages in the Sp configuration and no more than 4 non-chiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration can be optionally contiguous or non-contiguous. In some embodiments, the internucleotide linkages in the Rp configuration can be optionally contiguous or non-contiguous. In some embodiments, the non-chiral internucleotide linkages can be optionally contiguous or non-contiguous.

[0401] In some embodiments, the dsRNA molecules described herein comprise blocks that are stereochemical blocks. In some embodiments, the blocks are Rp blocks, where each internucleotide linkage of the block is an Rp. In some embodiments, the 5'-block is an Rp block. In some embodiments, the 3'-block is an Rp block. In some embodiments, the blocks are Sp blocks, where each internucleotide linkage of the block is an Sp. In some embodiments, the 5'-block is an Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, the provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, the provided oligonucleotides comprise one or more Rp blocks but no Sp blocks. In some embodiments, the provided oligonucleotides comprise one or more Sp blocks but no Rp blocks. In some embodiments, the provided oligonucleotides comprise one or more PO blocks, where each internucleotide linkage is a natural phosphate linkage.

[0402] In some embodiments, the dsRNA molecules described herein comprise a 5'-block that is an Sp block in which each sugar moiety comprises a 2'-fluoro modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-fluoro modification. In some embodiments, the 5'-block is an Sp block in which each internucleoside linkage is a phosphorothioate linkage and each sugar moiety comprises a 2'-fluoro modification. In some embodiments, the 5'-block comprises four or more nucleoside units. In some embodiments, the 5'-block comprises five or more nucleoside units. In some embodiments, the 5'-block comprises six or more nucleoside units. In some embodiments, the 5'-block comprises seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety comprises a 2'-fluoro modification. In some embodiments, the 3'-block is an Sp block, in which each of the internucleotide linkages is a modified internucleotide linkage and each sugar moiety comprises a 2'-fluoro modification. In some embodiments, the 3'-block is an Sp block, in which each of the internucleotide linkages is a phosphorothioate linkage and each sugar moiety comprises a 2'-fluoro modification. In some embodiments, the 3'-block comprises four or more nucleoside units. In some embodiments, the 3'-block comprises five or more nucleoside units. In some embodiments, the 3'-block comprises six or more nucleoside units. In some embodiments, the 3'-block comprises seven or more nucleoside units.

[0403] In some embodiments, the dsRNA molecules described herein comprise a certain type of nucleoside in a region or oligonucleotide, followed by a particular type of internucleotide linkage, such as a natural phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0404] A variety of publications describe multimeric siRNA, and all of them can be used for the oligonucleotide and dsRNA of the present invention.Such publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, and they are all incorporated herein.

[0405] In some embodiments, the dsRNA molecules described herein include one or more overhang regions and / or capping groups at the 3' or 5' end or both ends of the strand. The overhangs can be 1 to 10 nucleotides in length. For example, the overhangs can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the overhangs are 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being offset from one another. The overhangs can form mismatches with the target sequence, be complementary to the targeted gene sequence, or be other sequences. The first and second strands can also be joined, for example, by additional bases to form a hairpin, or by other non-basic linkers.

[0406] In some embodiments, the nucleotide in the overhang region of the dsRNA molecule described herein can be independently modified or unmodified nucleotide, for example, but not limited to, 2'-sugar modified, for example, 2'-fluoro, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine, 2'-O-methoxyethyl adenosine, 2'-O-methoxyethyl-5-methylcytidine, GNA, SNA, hGNA, hhGNA, mGNA, TNA, h'GNA, and any combination thereof.For example, dTdT can be the overhang sequence at either end of either strand.Overhang can form mismatch with target mRNA, or be complementary to the target gene sequence, or be other sequences.

[0407] The 5'-overhang or 3'-overhang of the sense strand, antisense strand, or both strands of the dsRNA molecule described herein can be phosphorylated.In some embodiments, the overhang region contains two nucleotides with phosphorothioate between them, and these two nucleotides can be the same or different.In some embodiments, the overhang is present at the 3' end of the sense strand, antisense strand, or both strands.In some embodiments, this 3'-overhang is present in the antisense strand.In some embodiments, this 3'-overhang is present in the sense strand.

[0408] The dsRNA molecule described herein may contain only one overhang, which can enhance its interference activity without affecting the overall stability of dsRNA.For example, the single-stranded overhang can be located at the 3' end of the sense strand or at the 3' end of the antisense strand.dsRNA can also have a blunt end, which is located at the 5' end of the antisense strand (or at the 3' end of the sense strand) or vice versa.

[0409] Generally, the antisense strand of dsRNA has a nucleotide overhang at its 3' end, and its 5' end is blunt.Without being bound by theory, the blunt end at the 5' end of the asymmetric antisense strand and the 3' end overhang of the antisense strand are advantageous for the loading process of guide strand into RISC.For example, the single overhang is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides long.In some embodiments, the dsRNA has a 2-nucleotide overhang at the 3' end of the antisense strand, and the blunt end at the 5' end of the antisense strand.

[0410] dsRNA described herein can comprise one or more modified nucleotides.For example, all nucleotides in the sense strand and antisense strand of dsRNA molecule can be modified.Each nucleotide can be modified with the same or different modifications, and these modifications can include one or more modifications of one or both of non-linked phosphate oxygen and / or one or more of linked phosphate oxygen; modification of ribose sugar components; replacement of ribose sugar; complete replacement of phosphate moiety with " dephosphorylation " linker; modification or replacement of natural base; and replacement or modification of ribose-phosphate backbone.

[0411] Because nucleic acids are polymers of subunits, many of the modifications occur at positions that are repeated within the nucleic acid, such as modifications of bases, or phosphate moieties, or non-linked Os in phosphate moieties. In some cases, modifications will occur at all of the target positions in the nucleic acid, but in many cases, this will not be the case. For example, modifications may occur only at the 3'-terminal position or the 5'-terminal position, or only in the central region, or only in the non-terminal region, or only in the terminal region, e.g., on the terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of the strand. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in the double-stranded region of the RNA or only in the single-stranded region of the RNA. For example, phosphorothioate modifications of non-linked O positions may be present only at one or both ends, or may be present only in terminal regions, e.g., positions on the terminal nucleotide, or in the last 2, 3, 4, 5, or 10 nucleotides of the strand, or may be present in double-stranded and single-stranded regions, particularly at the termini. One or more 5' ends may be phosphorylated.

[0412] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, for example, the 5' overhang or the 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, with the modifications described herein. Modifications can include, for example, the use of modifications at the 2' position of the ribose sugar, such as modifications known in the art, such as the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications in place of the ribose sugar of the nucleobase, and modifications in the phosphate group, such as phosphorothioate modifications. The overhang need not be homologous to the target sequence.

[0413] In some embodiments, the dsRNA molecules described herein contain alternating patterns of modifications, particularly in the B1, B2, B3, B1', B2', B3', and B4' regions. As used herein, the term "alternating motif" or "alternating pattern" refers to a motif having one or more modifications, each occurring on alternating nucleotides of a single strand. Alternating nucleotides can refer to patterns such as every other nucleotide or every third nucleotide. For example, if A, B, and C each represent a type of modification to a nucleotide, the alternating motif can be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AAABBBAAABBB...," or "ABCABCABCABC...," etc.

[0414] The types of modifications within the alternating motif can be the same or different. For example, if A, B, C, and D each represent a type of modification on a nucleotide, the alternation pattern, i.e., the modifications at every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possible modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0415] In some embodiments, the dsRNA molecules described herein contain a modification pattern for the alternating motif on the sense strand that is shifted relative to the modification pattern for the alternating motif on the antisense strand. The shift can be such that the modified groups of nucleotides in the sense strand correspond to different modified groups of nucleotides in the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can begin with "ABABAB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can begin with "BABABA" from 3' to 5' of the strand, within the duplex region. As another example, the alternating motif in the sense strand can begin with "AABBAABB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can begin with "BBAABBAA" from 3' to 5' of the strand, within the duplex region, such that a complete or partial shift in the modification pattern occurs between the sense and antisense strands.

[0416] In some embodiments of any one of the aspects described herein, at least one strand, e.g., both strands, of the oligonucleotides described herein or the dsRNAs described herein are 5'-phosphorylated or contain a phosphoryl analog at the 5' prime end. 5'-phosphate modifications include those compatible with RISC-mediated gene silencing. Suitable modifications include 5'-monophosphate ((HO)2(O)PO-5'), 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'), 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)- O-5'), 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5'-monothiophosphate (phosphorothioate, (HO)2(S)PO-5'), 5'-monodithiophosphate (phosphorodithioate, (HO)(HS)(S)PO-5'), 5'-phosphorothiolate phosphate ((HO)2(O)PS-5'); any further combination of oxygen / sulfur substituted monophosphates, diphosphates and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkyl phosphonates (e.g., R-P(OH)(O)-O-5'-, R = alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkenyl phosphonates (i.e., vinyl, substituted vinyl, e.g., OH)2(O)P-5'-CH= or (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonates (e.g., R(OH)(O)PO-5', R = alkyl ether, e.g., methoxymethyl (MeOCH2-), ethoxymethyl, etc.). Other exemplary 5'-modifications include those in which Z is alkyl, optionally substituted at least once, such as ((HO)2(X)PO[-(CH2)a -OP(X)(OH)-O] b -5', ((HO)2(X)PO[-(CH2) a -P(X)(OH)-O] b -5', ((HO)2(X)P-[-(CH2) a -OP(X)(OH)-O] b -5'; Dialkyl terminal phosphate and phosphate mimic: HO[-(CH2) a -OP(X)(OH)-O] b -5', H2N[-(CH2) a -OP(X)(OH)-O] b -5', H[-(CH2) a -OP(X)(OH)-O] b -5', Me2N[-(CH2) a -OP(X)(OH)-O] b -5', HO[-(CH2) a -P(X)(OH)-O] b -5', H2N[-(CH2) a -P(X)(OH)-O] b -5', H[-(CH2) a -P(X)(OH)-O] b -5', Me2N[-(CH2) a -P(X)(OH)-O] b -5', where a and b are each independently 1 to 10. Another embodiment is BH3, BH3 - and / or containing substitution of oxygen and / or sulfur by Se.

[0417] In some embodiments of any one of the aspects described herein, at least one (for example, both) strands of the oligonucleotide described herein or the dsRNA described herein comprise a 5'-vinyl phosphonate group.For example, at least one (for example, both) strands of the oligonucleotide described herein or the dsRNA described herein comprise a 5'-E-vinyl or at least one (for example, both) strands of the dsRNA described herein comprise a 5'-Z-vinyl phosphonate group.In some other non-limiting examples, the oligonucleotide comprises a 5'-Z-vinyl phosphonate group.

[0418] In one example, the 5'-modification can be placed in the antisense strand of a double-stranded nucleic acid, such as a dsRNA molecule. For example, the antisense strand comprises a 5'-E-vinyl phosphonate. In some other non-limiting examples, the antisense strand comprises a 5'-Z-vinyl phosphonate group.

[0419] In some embodiments, the sense strand comprises a 5'-morpholino, 5'-dimethylamino, 5'-deoxy, inverted abasic, or inverted abasic locked nucleic acid modification at the 5' end.

[0420] The dsRNA agents of the present invention can include a thermodestabilizing modification in the seed region of the antisense strand (i.e., positions 2-9 from the 5' end of the antisense strand) to reduce or inhibit off-target gene silencing. Without wishing to be bound by theory, dsRNAs having an antisense strand containing at least one duplex thermodestabilizing modification within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand contains at least one (e.g., one, two, three, four, five, or more) duplex thermodestabilizing modification within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, the duplex thermodestabilizing modification is located at positions 2-9, or preferably positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the duplex thermodestabilizing modification is located at positions 5, 6, 7, or 8 from the 5' end of the antisense strand.

[0421] In some further embodiments, the thermodestabilizing modification of the duplex is located at position 7 from the 5' end of the antisense strand.

[0422] The term "thermally destabilizing modification" encompasses modifications that result in a dsRNA with a reduced overall melting temperature (Tm), preferably a Tm that is 1, 2, 3, or 4 degrees lower than the Tm of a dsRNA lacking such modification. In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5, 6, 7, 8, or 9 from the 5' end of the antisense strand.

[0423] Thermolabile modifications can include, but are not limited to, abasic modifications; mismatches with opposing nucleotides in opposing strands; and sugar modifications such as 2'-deoxy modifications or acyclic nucleotides, e.g., unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs). For example, thermolabile modifications can include, but are not limited to, the following mUNA and GNA building blocks: TIFF2024539093000031.tif239166TIFF2024539093000032.tif170164.

[0424] In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA.

[0425] In some embodiments, the destabilizing modified mUNA is TIFF2024539093000033.tif87152R = H, OH;OMe;Cl, F;OH;O-(CH2)2OMe;SMe, NMe2;NH2;Me;CCH(alkyne), O-nPr;O-alkyl;O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles and the stereochemistry is R or S and combinations of R and S for unspecified chiral centers.

[0426] In some embodiments, the destabilizing modified mUNA is TIFF2024539093000034.tif53147R = H, OH;OMe;Cl, F;OH;O-(CH2)2OMe;SMe, NMe2;NH2;Me;CCH(alkyne), O-nPr;O-alkyl;O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles and the stereochemistry is R or S and combinations of R and S for unspecified chiral centers.

[0427] In some embodiments, the destabilizing modified mUNA is TIFF2024539093000035.tif85156R=H, OMe;F;OH;O-(CH2)2OMe;SMe, NMe2;NH2;Me;O-nPr;O-alkyl;O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard monocyclic, bicyclic, and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 7-deazapurine and the stereochemistry is R or S and combinations of R and S for unspecified chiral centers.

[0428] In some embodiments, the destabilizing modified mUNA is TIFF2024539093000036.tif86151R = H, OH;OMe;Cl, F;OH;O-(CH2)2OMe;SMe, NMe2;NH2;Me;CCH(alkyne), O-nPr;O-alkyl;O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles selected from the group consisting of The stereochemistry is R or S and combinations of R and S for unspecified chiral centers.

[0429] In some embodiments, the destabilizing modified mUNA is TIFF2024539093000037.tif51147R = H, OH;OMe;Cl, F;OH;O-(CH2)2OMe;SMe, NMe2;NH2;Me;CCH(alkyne), O-nPr;O-alkyl;O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles and the stereochemistry is R or S and combinations of R and S for unspecified chiral centers.

[0430] In some embodiments, the modified mUNA is TIFF2024539093000038.tif84157R=H, OMe;F;OH;O-(CH2)2OMe;SMe, NMe2;NH2;Me;O-nPr;O-alkyl;O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard monocyclic, bicyclic, and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 7-deazapurine and the stereochemistry is R or S and combinations of R and S for unspecified chiral centers.

[0431] Exemplary abasic modifications include, but are not limited to, the following modifications: TIFF2024539093000039.tif60128 Where R = H, Me, Et or OMe, R' = H, Me, Et or OMe, R" = H, Me, Et or OMe TIFF2024539093000040.tif45135 where B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic.

[0432] Exemplary sugar modifications include, but are not limited to, the following modifications: TIFF2024539093000041.tif81132 where B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic.

[0433] In some embodiments, the thermally destabilizing modification of the duplex is selected from the mUNA and GNA building blocks described herein in Examples 1-3. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, and 2'-mUNA. In some further embodiments thereof, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA, and h'GNA (Mod A through Mod K).

[0434] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, one in which any of the bonds between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') are absent, and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is missing from the nucleotide. In some embodiments, an acyclic nucleotide is TIFF2024539093000042.tif35155, where B is a modified or unmodified nucleobase, R1 and R2 are independently H, halogen, OR3, or alkyl, and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses a monomer in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) is removed (see Mikhailov et al., Tetrahedron Letters, 26(17):2059 (1985) and Fluiter et al., Mol. Biosyst., 10:1039 (2009), which are incorporated herein by reference in their entireties). Acyclic derivatives increase backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked by 2'-5' or 3'-5' linkages.

[0435] The term "GNA" refers to glycol nucleic acid, a polymer that is similar to DNA or RNA but differs in that its "backbone" composition consists of repeating glycerol units linked by phosphodiester bonds: Points to TIFF2024539093000043.tif57128.

[0436] The thermally destabilizing modification of the duplex can be a mismatch (i.e., non-complementary base pair) between the thermally destabilizing nucleotide in the dsRNA duplex and the opposite nucleotide in the opposite strand.Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof.Other mismatch base pairings known in the art can also be applied to the present invention.Mismatches can occur between nucleotides that are either natural or modified nucleotides.That is, mismatch base pairing can occur between the nucleobases of each nucleotide, regardless of the modification on the ribose sugar of the nucleotide.In certain embodiments, the dsRNA molecule contains at least one nucleobase that is a 2'-deoxynucleobase in mismatch pairing, for example, the 2'-deoxynucleobase is in the sense strand.

[0437] In some embodiments, the duplex thermodestabilizing modification in the seed region of the antisense strand is a nucleotide that has impaired WCH binding to a complementary base on the target mRNA, e.g., Includes TIFF2024539093000044.tif72135.

[0438] Further examples of abasic nucleotide modifications, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are detailed in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0439] Thermally destabilizing modifications may also include universal bases that have reduced or eliminated ability to hydrogen bond with opposing bases, and phosphate modifications.

[0440] In some embodiments, the thermal destabilizing modification of duplex comprises the nucleotide with non-standard base, for example but not limited to, the nucleobase modification that the ability to form hydrogen bond with the base in opposite strand is impaired or completely lost.These nucleobase modifications have been evaluated for the destabilization of the central region of dsRNA duplex, as described in WO 2010 / 0011895, and this document is incorporated herein by reference in its entirety.Exemplary nucleobase modifications include: The file is TIFF2024539093000045.tif63147.

[0441] In some embodiments, the duplex thermodestabilizing modification in the seed region of the antisense strand is one or more α-nucleotides complementary to bases on the target mRNA, e.g., TIFF2024539093000046.tif20146, where R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.

[0442] Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes relative to natural phosphodiester linkages include: The file is TIFF2024539093000047.tif30139.

[0443] The alkyl R group can be a C1-C6 alkyl. Specific alkyl R groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0444] It should be noted that the thermodestabilizing modification can replace 2'-deoxynucleotides in the antisense strand. For example, the 2'-deoxynucleotides at positions 2, 5, 7, 12, 14, and / or 16, counting from the 5' end of the antisense strand, can be replaced with the thermodestabilizing modifications described herein. Thus, in some embodiments, the antisense strand comprises thermodestabilizing modifications at 1, 2, 3, 4, 5, and / or 6 of positions 2, 5, 7, 12, 14, and / or 16, counting from the 5' end of the antisense strand. For example, the antisense strand comprises thermodestabilizing modifications at positions 5 and 7, counting from the 5' end of the antisense strand.

[0445] In addition to the antisense strand containing thermal destabilizing modifications, dsRNA can also contain one or more stabilizing modifications.For example, dsRNA can contain at least two (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications.Without being limited, all stabilizing modifications can be present in one strand.In some embodiments, both the sense strand and the antisense strand contain at least two stabilizing modifications.The stabilizing modifications can be present on any nucleotide of the sense strand or the antisense strand.For example, the stabilizing modifications can be present on every nucleotide of the sense strand and / or the antisense strand, or each stabilizing modification can be present in an alternating pattern on the sense strand or the antisense strand, or the sense strand or the antisense strand contains both stabilizing modifications in an alternating pattern.The alternating pattern of the stabilizing modifications on the sense strand can be the same or different from that of the antisense strand, and the alternating pattern of the stabilizing modifications on the sense strand can be shifted relative to the alternating pattern of the stabilizing modifications on the antisense strand.

[0446] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications. Without limitation, the stabilizing modifications in the antisense strand can be located at any position. In some embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5' end. In some further embodiments, the antisense strand comprises stabilizing modifications at positions 2, 14, and 16 from the 5' end.

[0447] In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification.For example, the stabilizing modification can be the nucleotide at the 5' or 3' end of the destabilizing modification, i.e., the nucleotide at the -1 or +1 position from the position of the destabilizing modification.In some embodiments, the antisense strand comprises the stabilizing modification at the 5' end and 3' end of the destabilizing modification, i.e., the nucleotide at the -1 position and +1 position from the position of the destabilizing modification.

[0448] In some embodiments, the antisense strand contains at least two stabilizing modifications at the 3' end of the destabilizing modification, i.e., at least two stabilizing modifications at positions +1 and +2 from the position of the destabilizing modification. In some embodiments, the sense strand contains at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) stabilizing modifications. Without limitation, the stabilizing modifications in the sense strand can be located at any position. In some embodiments, the sense strand contains stabilizing modifications at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand contains stabilizing modifications at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand contains stabilizing modifications at positions 11, 12, and 15 of the antisense strand, relative to or complementary to positions 11, 12, 13, and 15 of the antisense strand, relative to the 5' end of the antisense strand. In some other embodiments, the sense strand contains stabilizing modifications at positions 11, 12, 13, and 15 of the antisense strand, relative to or complementary to positions 11, 12, 13, and 15 of the antisense strand, relative to the 5' end of the antisense strand. In some embodiments, the sense strand comprises blocks of two, three, or four stabilizing modifications.

[0449] In some embodiments, the sense strand does not contain a stabilizing modification at a position opposite or complementary to a thermodestabilizing modification of the duplex in the antisense strand.

[0450] Exemplary thermostabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermostabilizing modifications include, but are not limited to, LNA.

[0451] Note that the thermostabilizing modification can replace 2'-fluoro nucleotides in the sense strand and / or antisense strand. For example, the 2'-fluoro nucleotides at positions 8, 9, 10, 11, and / or 12, counting from the 5' end of the sense strand, can be replaced with a thermostabilizing modification. Similarly, the 2'-fluoro nucleotide at position 14, counting from the 5' end of the antisense strand, can be replaced with a thermostabilizing modification.

[0452] In order for dsRNA molecules to be more effective in vivo, antisense strand must have some metabolic stability.In other words, in order for dsRNA molecules to be more effective in vivo, after administration, after a certain period, some amount of antisense strand may need to exist in vivo.Therefore, in some embodiments, after in vivo administration, on the 5th day, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver.In some embodiments, after in vivo administration, on the 6th day, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver. In some embodiments, after in vivo administration on the 7th day, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA is present in vivo, for example in mouse liver.In some embodiments, after in vivo administration on the 8th day, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA is present in vivo, for example in mouse liver.In some embodiments, after in vivo administration on the 9th day, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA is present in vivo, for example in mouse liver. In some embodiments, 10 days after in vivo administration, at least 40%, e.g., at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, e.g., in the mouse liver.In some embodiments, after in vivo administration on the 11th day, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%.In some embodiments, after in vivo administration on the 12th day, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%.In some embodiments, after in vivo administration on the 13th day, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%. In some embodiments, after in vivo administration, 14 days later, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%.In some embodiments, after in vivo administration, 15 days later, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%.

[0453] Ligand Embodiments of the various aspects described herein include ligands. Without wishing to be bound by theory, ligands alter one or more properties of the attached molecule (e.g., the oligonucleotides described herein), including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, intracellular distribution, cellular uptake, charge, and clearance. Ligands are routinely used in chemistry and are linked to the parent compound either directly or via an optional linking moiety or group. A preferred list of ligands includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0454] Preferred ligands applicable to the present invention include lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49); phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0455] Ligands can include naturally occurring molecules or recombinant or synthetic molecules. Exemplary ligands include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, polyphosphazine, polyethyleneimine, cationic groups, spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, and quaternary polyamines. Salts, thyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucins, glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamates, polyaspartates, aptamers, asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalating agents (e.g., acridines), crosslinkers (e.g., psoralens, mitomas), isin C), porphyrins (e.g., TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., steroids, bile acids, cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., alpha helical peptides, amphipathic peptides, RGD peptides, cell penetrating peptides, endosomolytic / fusogenic peptides), alkylating agents, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., naproxen), Sen, aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, polyvalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin, and pyridoxal), vitamin cofactors, lipopolysaccharide, p38 These include, but are not limited to, activators of MAP kinase, activators of NF-κB, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, gamma interferon, natural or recombinant low-density lipoprotein (LDL), natural or recombinant high-density lipoprotein (HDL), and cell-permeation agents (e.g., a.helical cell-permeation agents).

[0456] Peptide and peptidomimetic ligands include natural or modified peptides, such as D- or L-peptides; α, β, or γ peptides; N-methylpeptides; azapeptides; peptides with one or more amide linkages, i.e., peptides in which a linkage is replaced with one or more urea, thiourea, carbamate, or sulfonylurea linkages; or cyclic peptides. Peptide mimetics (also referred to herein as oligopeptide mimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Peptide or peptidomimetic ligands can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0457] Exemplary amphipathic peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, esculentinis-1, and caerin.

[0458] As used herein, the term "endosomolytic ligand" refers to a molecule having endosomolytic properties. An endosomolytic ligand promotes the lysis of a cellular compartment, such as an endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other vesicular bodies within a cell, and / or the transport of a composition of the invention or a component thereof therefrom into the cytoplasm of a cell. Exemplary endosomolytic ligands include, but are not limited to, imidazoles, poly- or oligoimidazoles, linear or branched polyethyleneimines (PEI), linear and branched polyamines such as spermine, cationic linear and branched polyamines, polycarboxylates, polycations, shielded oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, linear or branched polymers with shielded or unshielded cationic or anionic charges, dendrimers with shielded or unshielded cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.

[0459] Exemplary endosomolytic / fusogenic peptides include: Examples include, but are not limited to, TIFF2024539093000048.tif92160.

[0460] Without wishing to be bound by theory, fusogenic lipids fuse with membranes, thereby destabilizing them. Fusogenic lipids typically have small head groups and unsaturated acyl chains. Exemplary fusogenic lipids include, but are not limited to, 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (also referred to herein as XTC).

[0461] Synthetic polymers with endosomolytic activity that can be used in the present invention are described in U.S. Patent Application Publication Nos. 2009 / 0048410, 2009 / 0023890, 2008 / 0287630, 2008 / 0287628, 2008 / 0281044, 2008 / 0281041, 2008 / 0269450, 2007 / 0105804, 20070036865, and 2004 / 0198687, the contents of which are incorporated herein by reference in their entireties.

[0462] Exemplary cell-penetrating peptides include: Examples include, but are not limited to, TIFF2024539093000049.tif85160.

[0463] Exemplary cationic groups include protonated amino groups, such as O-amine (amine = NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); aminoalkoxy, such as O(CH)n Amines (e.g., amine = NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino), amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid), and NH(CHCHNH) n Examples include, but are not limited to, those derived from CH2CH2-amine (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino).

[0464] As used herein, the term "targeting ligand" refers to any molecule that provides enhanced affinity to a selected target, such as a cell, cell type, tissue, organ, body region, or compartment, such as a cell, tissue, or organ compartment.Exemplary targeting ligands include, but are not limited to, antibodies, antigens, folate, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.

[0465] Carbohydrate-based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, such as GalNAc2 and GalNAc3, D-mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, multivalent fucose, glycosylated polyamino acids, and lectins.The term multivalent indicates the presence of two or more monosaccharide units.Such monosaccharide subunits can be linked to each other by glycosidic bonds or linked to scaffold molecules.

[0466] Some of the folates and folic acid analogs applicable to the present invention are described in U.S. Pat. Nos. 2,816,110, 5,552,545, 6,335,434, and 7,128,893, the contents of which are incorporated herein by reference in their enti...

Claims

1. A double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand complementary to the sense strand, the dsRNA has a double-stranded region of at least about 15 base pairs; the antisense strand comprising (a) and (b): (a) a 5-terminal nucleoside which is a 2'-geminal substituted nucleoside of formula (II) or (II'): and (b) at least one of positions 2 to 9 (e.g., positions 2, 3, 4, 5, 6, 7, 8, and / or 9) counting from the 5' end of the antisense strand, a 2'-geminal substituted nucleoside of formula (I) or (I'): and In formulas (I), (I'), (II) and (II'), X is O, S, C (R X ) 2 , or N(R XN ) and Each R X are independently hydrogen, halogen, or optionally substituted C 1～4 Alkyl, C 1～4 Haloalkyl, optionally substituted C 2～4 Alkenyl, or optionally substituted C 2～4 alkynyl, or both R X Overall, =O, =S, =N (R N ), or =CH 2 Forming R XN is hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 1 ~C 30 Alkoxy, C 1～4 Haloalkyl, optionally substituted C 2～4 Alkenyl, optionally substituted C 2～4 Alkynyl, optionally substituted C 1～30 Alkyl-CO 2 H, or a nitrogen protecting group, B is an optionally modified nucleobase; R a' is halogen, hydrogen, -OR a2 , -SR a3 , optionally substituted C 1～30 Alkyl, C 1～30 Haloalkyl, optionally substituted C 2～30 Alkenyl, optionally substituted C 2～30 Alkynyl, or optionally substituted C 1～30 Alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O) m CH 2 CH 2 OR a4 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH) n CH 2 CH 2 -R a5 , NHC(O)R a4 , to a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, a solid support, a linker covalently attached to a solid support, or an internucleoside linkage to a subsequent nucleoside; R a2 is a hydrogen or hydroxyl protecting group, R a3 is a hydrogen or sulfur protecting group, R a4 independently for each occurrence, H, C 1 ~C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R a5 and R a5 independently for each occurrence, an amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino; m is 1 to 50; n is 1 to 50; R b may be substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, optionally substituted C 2～30 alkynyl, or halogen; R c is the bond to the internucleoside linkage to the subsequent nucleoside, hydrogen, halogen, -OR c2 , -SR c3 , optionally substituted C 1～30 Alkyl, C 1～30 Haloalkyl, optionally substituted C 2～30 Alkenyl, optionally substituted C 2～30 Alkynyl, or optionally substituted C 1～30 Alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O) r CH 2 CH 2 OR c4 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH) s CH2CH2-R c5 , NHC(O)R c4 , a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, a solid support, or a linker covalently attached to a solid support, wherein optionally at least R c or R a' is the bond to the internucleoside linkage to the subsequent nucleoside, R c2 is a hydrogen or hydroxyl protecting group, R c3 is a hydrogen or sulfur protecting group, R c4 independently for each occurrence, H, C 1 ~C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R c5 and R c5 independently for each occurrence, an amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino; r is 1 to 50; s is 1 to 50, R 4 is hydrogen, optionally substituted C 1～6 Alkyl, optionally substituted C 2～6 Alkenyl, optionally substituted C 2～6 Alkynyl, or optionally substituted C 1～6 Is it an alkoxy? Alternatively, R 4 and R a is, as a whole, 4'-C(R a11 R a12 ) v -Y-2' or 4'-YC (R a11 R a12 ) v -2', Y is -O-, -CH 2 -, -CH(Me)-, -C(CH 3 ) 2 -, -S-, -N (R a13 )-, -C(O)-, -C(S)-, -S(O)-, -S(O) 2 -, -OC(O)-, -C(O)O-, -N(R a13 )C(O)-, or -C(O)N(R a13 ) - and R a11 and R a12 are independently H, optionally substituted C 1 ~C 6 Alkyl, optionally substituted C 2 ~C 6 Alkenyl or optionally substituted C 2 ~C 6 is alkynyl, R a13 is hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 1 ~C 30 Alkoxy, C 1～4 Haloalkyl, optionally substituted C 2～4 Alkenyl, optionally substituted C 2～4 Alkynyl, optionally substituted C 1～30 Alkyl-CO 2 H, or a nitrogen protecting group, v is 1, 2 or 3, Alternatively, R 4 and R c C, together with the atoms to which they are attached, may be substituted 3～8 Cycloalkyl, optionally substituted C 3～8 forming a cycloalkenyl or an optionally substituted 3- to 8-membered heterocyclyl, R d is -CH(R d1 )-R d2 or -C(R d1 ) = CHR d2 and R d1 is hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted -C 2～30 alkenyl, or optionally substituted -C 2～30 is alkynyl, R d2 is the bond to the internucleoside linkage to the preceding nucleoside, R e is optionally substituted -C 2～6 Alkenyl-R e1 , optionally substituted C 1～6 Alkyl-R e1 , or optionally substituted -C 2～6 Alkynyl-R e1 and R e1 は、-P(O)(OR e4 ) 2 、-OR e2 、-SR e3 、-P(S)(OR e4 ) 2 、-P(S)(SR e5 )(OR e4 )、-P(S)(SR e5 ) 2 、-OP(O)(OR e4 ) 2 、-OP(S)(OR e4 ) 2 、-OP(S)(SR e5 )(OR e4 )、-OP(S)(SR e5 ) 2 、-SP(O)(OR e4 ) 2 、-SP(S)(OR e4 ) 2 、-SP(S)(SR e5 )(OR e4 )、または-SP(S)(SR e5 ) 2 であり、 R e2 is a hydrogen or oxygen protecting group, R e3 is a hydrogen or sulfur protecting group, Each R e4 are independently hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, or optionally substituted C 2～30 alkynyl, or oxygen protecting group; and each R e5 are independently hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, or optionally substituted C 2～30 alkynyl, or sulfur protecting group, The dsRNA.

2. (i) The 5'-terminal nucleotide of the antisense strand is a 2'-geminal substituted nucleotide of formula (II). (ii) the antisense strand contains a vinyl phosphonate (e.g., E-vinyl phosphonate) group at its 5' end; (iii) the 5'-terminal nucleotide of the antisense strand is a 2'-geminally substituted nucleotide of formula (II), wherein R e is a vinyl phosphonate (e.g., R e is -CH=CHR e1 and R e1 -P(O)(OR e4 ) 2 ) and / or (iv) the antisense strand comprises a nucleoside of formula (I) at least at position 3, at least at position 4, at least at position 5, at least at position 6, at least at position 7, at least at position 8, and / or at least at position 9, counting from the 5' end of the antisense strand; The dsRNA of claim 1.

3. (A) (i) The 2'-geminal substituted nucleoside of formula (II) is a nucleoside of formula (IIA): or formula (IIB): or or (ii) the 2'-geminal substituted nucleoside of formula (II') is a nucleoside of formula (IIA'): Or formula (IIB'): and / or (B) (i) The 2'-geminal substituted nucleoside of formula (I) is represented by formula (IA): or formula (IB): or or (ii) the 2'-geminal substituted nucleoside of formula (I') is a nucleoside of formula (IA'): or formula (IB'): Follow The dsRNA of claim 1.

4. (i) X is O, (ii) (A) R a' But hydrogen, halogen, -OR a2 , optionally substituted C 1 ~C 30 Alkyl, optionally substituted C 1 ~C 30 Alkoxy, -O(CH 2 CH 2 O) m CH 2 CH 2 OR a4 , or -NH(CH 2 CH 2 NH) n CH 2 CH 2 -R a5 Or (B) R a' But halogen, hydrogen, -OR a2 , or optionally substituted C 1 ~C 30 Is it an alkoxy? (C) R a' But halogen, -OR a2 , or optionally substituted C 1 ~C 30 Is it an alkoxy? (D) R a' is F, Cl, OH or optionally substituted C 1 ~C 30 is alkoxy, or (E) R a' But amino or C 1 ~C 6 optionally substituted with alkoxy, C 1 ~C 30 is an alkoxy, (iii) (A) R b may be substituted C 1～6 Alkyl, C 1～6 Haloalkyl, optionally substituted C 2～6 Alkenyl, optionally substituted C 2～6 alkynyl or halogen; (B) R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl or propargyl, or (C) R b is methyl, vinyl, ethynyl, allyl or propargyl; (iv) (A) R 4 is hydrogen, optionally substituted C 1～6 Alkyl, optionally substituted C 2～6 Alkenyl, optionally substituted C 2～6 Alkynyl, or optionally substituted C 1～6 is alkoxy, or (B) R 4 is hydrogen, (v) R d -CH(R d1 )-X d -R d2 That is, (vi) X d is O, (vii)R d1 is hydrogen or optionally substituted C 1 ~C 6 Is it alkyl? (viii) R e Ga-C 2～6 Alkenyl-R e1 or C 1～6 Alkyl-R e1 and C 1～6 Alkyl and C 2～6 Alkenyl may be optionally substituted; (ix) R e -CH=CHR e1 That is, (x)R e1 -P(O)(OR e4 ) 2 , -OR e2 OR -OP (O) (OR e4 ) 2 and / or (xi) R e2 is hydrogen or optionally substituted C 1 ~C 6 is alkyl, The dsRNA of claim 1.

5. a 2'-geminal substituted nucleoside of formula (I) or (I') at least one of positions 2, 3, 4, 5, 6, 7, 8, 9, or 10, counting from the 5' end of the antisense strand; and optionally the 2'-geminal substituted nucleoside of formula (I) is at position 7, counting from the 5' end of the antisense strand; The dsRNA of claim 1. (i) the antisense strand further comprises at least one modified internucleoside linkage; (ii) the antisense strand further comprises at least one modified nucleobase; and / or (iii) The dsRNA of claim 1, wherein the antisense strand further comprises at least one nucleoside modified at the 2' position, and the nucleotide modified at the 2' position is not a 2'-geminal nucleoside. (i) The 2'-geminal substituted nucleoside of formula (II) is a 2'-geminal substituted nucleoside of formula (IIA): and During the ceremony, X is O, R a' is halogen (e.g., F, Cl, or Br), hydroxyl, optionally substituted C 1～30 Alkoxy (e.g., -(CH2 2 ) n CH 3 where n is 1 to 21, e.g., 1, 16, or -(CH 2 ) m -NH 2 where m is 2 to 10, for example 3 or 6, or -(CH 2 ) p -OMe, where p is 1-21, e.g., 1 or 2), or the bond to the internucleoside linkage to a subsequent nucleoside; R b may be substituted C 1～6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl) or halogen (e.g., F, Cl, or Br); R c is the bond or hydroxyl to the internucleoside linkage to the subsequent nucleoside, provided that R a' and R c is the bond to the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R e is -CH=CHR e1 where R e1 -P(O)(OR e4 ) 2 That is, (ii) The 2'-geminal substituted nucleoside of formula (II) is a 2'-geminal substituted nucleoside of formula (IIB): and During the ceremony, X is O, R a' is halogen (e.g., F, Cl, or Br), hydroxyl, optionally substituted C 1～30 Alkoxy (e.g., -(CH2 2 ) n CH 3 where n is 1 to 21, e.g., 1, 16, or -(CH 2 ) m -NH 2 where m is 2 to 10, for example 3 or 6, or -(CH 2 ) p -OMe, where p is 1-21, e.g., 1 or 2), or the bond to the internucleoside linkage to a subsequent nucleoside; R b may be substituted C 1～6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl) or halogen (e.g., F, Cl, or Br); R c is the bond or hydroxyl to the internucleoside linkage to the subsequent nucleoside, provided that R a' and R c is the bond to the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R e is -CH=CHR e1 where R e1 -P(O)(OR e4 ) 2 That is, (iii) The 2'-geminal substituted nucleoside of formula (I) is a 2'-geminal substituted nucleoside of formula (IA): and During the ceremony, X is O, R a' is halogen (e.g., F, Cl, or Br), hydroxyl, optionally substituted C 1～30 Alkoxy (e.g., -(CH2 2 ) n CH 3 where n is 1 to 21, e.g., 1, 16, or -(CH 2 ) m -NH 2 where m is 2 to 10, for example 3 or 6, or -(CH 2 ) p -OMe, where p is 1-21, e.g., 1 or 2), or the bond to the internucleoside linkage to a subsequent nucleoside; R b may be substituted C 1～6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl) or halogen (e.g., F, Cl, or Br); R c is the bond or hydroxyl to the internucleoside linkage to the subsequent nucleoside, provided that R a' and R c is the bond to the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R d is the bond to the internucleoside linkage to the preceding nucleoside, However, R a' and R c only one of which is the bond to the internucleoside linkage to the subsequent nucleoside, (iv) The 2'-geminal substituted nucleoside of formula (I) is a 2'-geminal substituted nucleoside of formula (IB): and During the ceremony, X is O, R a' is halogen (e.g., F, Cl, or Br), hydroxyl, optionally substituted C 1～30 Alkoxy (e.g., -(CH2 2 ) n CH 3 where n is 1 to 21, e.g., 1, 16, or -(CH 2 ) m -NH 2 where m is 2 to 10, for example 3 or 6, or -(CH 2 ) p -OMe, where p is 1-21, e.g., 1 or 2), or the bond to the internucleoside linkage to a subsequent nucleoside; R b may be substituted C 1～6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl) or halogen (e.g., F, Cl, or Br); R c is the bond or hydroxyl to the internucleoside linkage to the subsequent nucleoside, provided that R a' and R c is the bond to the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R d is the bond to the internucleoside linkage to the preceding nucleoside, However, R a' and R c only one of which is the bond to the internucleoside linkage to the subsequent nucleoside, The dsRNA of claim 1. (i) the antisense strand is at least about 17, or about 17 to 42 nucleotides in length; (ii) the antisense strand is about 19, about 20, about 21, about 22, about 23, about 24, about 25, or about 26 nucleotides in length, and optionally the antisense strand is about 22, about 23, about 24, or about 25 nucleotides in length; (iii) the sense strand is at least about 15, or about 15-40 nucleotides in length; (iv) the sense strand is about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length; and optionally the sense strand is about 21 nucleotides in length; (v) (a) the sense strand is 15 nucleotides in length and the antisense strand is 18, 19, 20, 21, or 22 (e.g., 20) nucleotides in length; (b) the sense strand is 19 nucleotides in length and the antisense strand is 19, 20, or 21 nucleotides in length; (c) the sense strand is 20 nucleotides in length and the antisense strand is 20, 21, or 22 nucleotides in length; (d) the sense strand is 21 nucleotides in length and the antisense strand is 21, 22, or 23 nucleotides in length; or (e) the sense strand is 20-24 (e.g., 22) nucleotides in length and the antisense strand is 34-38 (e.g., 36) nucleotides in length; (vi) having a double-stranded region of at least about 15, e.g., about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 or more base pairs, and optionally having a double-stranded region of about 21 base pairs; (vii) the sense strand is about 21 nucleotides in length, the antisense strand is about 21, about 22, about 23, about 24, or about 25 nucleotides in length, and the dsRNA comprises a double-stranded region of at least 18, e.g., 19, 20, or 21 base pairs; (viii) comprises at least one single-stranded overhang comprising 1 to 5 nucleotides (e.g., 1 or 2 nucleotides); (ix) the antisense strand comprises a single-stranded overhang at its 3' end; (x) comprises at least one blunt end, and optionally, the antisense strand comprises a blunt end at its 5' end; (xi) the antisense strand comprises a single-stranded overhang at its 3' end and a blunt end at its 5' end; (xii) at least four phosphorothioate internucleoside linkages, e.g., at least six phosphorothioate internucleoside linkages or at least eight phosphorothioate or at least ten phosphorothioate internucleoside linkages; (xiii) the antisense strand comprises at least two, e.g., three, four, six, or more, phosphorothioate internucleoside linkages, and optionally (A) the antisense strand comprises a phosphorothioate internucleoside linkage between positions 1 and 2, counting from the 3' end of the strand, and a phosphorothioate internucleoside linkage between positions 1 and 2, counting from the 5' end of the strand; or (B) the antisense strand contains a phosphorothioate internucleoside linkage between positions 1 and 2 and between positions 2 and 3, counting from the 3' end of the strand, and a phosphorothioate internucleoside linkage between positions 1 and 2, counting from the 5' end of the strand; or (C) the antisense strand contains phosphorothioate internucleoside linkages between positions 1 and 2 and between positions 2 and 3, counting from the 3' end of the strand, and phosphorothioate internucleoside linkages between positions 1 and 2 and between positions 2 and 3, counting from the 5' end of the strand; or (D) the antisense strand contains phosphorothioate internucleoside linkages between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, counting from the 3' end of the strand, and phosphorothioate internucleoside linkages between positions 1 and 2, counting from the 5' end of the strand; or (E) the antisense strand contains a phosphorothioate internucleoside linkage between positions 1 and 2, counting from the 3' end of the strand, and a phosphorothioate internucleoside linkage between positions 1 and 2 and between positions 2 and 3, counting from the 5' end of the strand; or (F) the antisense strand contains a phosphorothioate internucleoside linkage between positions 1 and 2, counting from the 3' end of the strand, and a phosphorothioate internucleoside linkage between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, counting from the 5' end of the strand; (xiv) the sense strand contains at least one, e.g., two, three, four, or more, phosphorothioate internucleoside linkages, and optionally (A) the sense strand contains a phosphorothioate internucleoside linkage between positions 1 and 2, counting from the 5' end of the strand; or (B) the sense strand contains phosphorothioate internucleoside linkages between positions 1 and 2, counting from the 5' end of the strand, and between positions 1 and 2, counting from the 3' end of the strand; or (C) the sense strand contains phosphorothioate internucleoside linkages between positions 1 and 2 and between positions 2 and 3, counting from the 5' end of the strand; or (D) the sense strand contains phosphorothioate internucleoside linkages between positions 1 and 2 and between positions 2 and 3, counting from the 5' end of the strand, and between positions 1 and 2 and between positions 2 and 3, counting from the 3' end of the strand; and / or (xv) a ligand, optionally comprising: (A) The ligand is linked to the sense strand; (B) The ligand is linked to the 3' end of the sense strand, or the ligand is linked to the 5' end of the sense strand; (C) the ligand is selected from the group consisting of peptides, centrins, antibodies (e.g., anti-CD4 antibodies and anti-CD117 antibodies), antibody fragments, T cell targeting ligands, B cell targeting ligands, cancer cell targeting ligands (e.g., DUPA, folate, and RGD), spleen targeting functionality, lung targeting functionality, bone marrow targeting functionality, phage display peptides, cell penetrating peptides (CPPs), integrin ligands, polyanionic ligands, polycationic ligands, monovalent and polyvalent carbohydrates (e.g., GalNAc, mannose, mannose-6 phosphate, mucose, and mulucose), kidney targeting ligands, BBB penetrating ligands, lipids, and amino acids (e.g., L-amino acids, D-amino acids, and β-amino acids); (D) the ligand contains GalNAc, and / or (E) The ligand is That is, The dsRNA of claim 1. (i) comprising at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 2'-fluoronucleotide; (ii) the antisense strand comprises at least one, e.g., 2, 3, 4, 5, or more, 2'-fluoronucleotide, and optionally: (A) the antisense strand contains 2'-fluoro nucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense strand; (B) the antisense strand contains 2'-fluoro nucleotides at positions 2, 6, 14, and 16, counting from the 5' end of the antisense strand; (C) the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 9, 14, and 16, counting from the 5' end of the antisense strand; or (D) the antisense strand contains 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the antisense strand; (iii) the antisense strand comprises at least one, e.g., 2, 3, 4, 5, or more, 2'-fluoronucleotide, and optionally (A) the sense strand contains 2'-fluoronucleotides at positions 7, 9, and 11, counting from the 5' end of the sense strand, or at positions 11, 13, and 15, counting from the 3' end of the sense strand; (B) the sense strand contains 2'-fluoronucleotides at positions 7, 9, 10, and 11, counting from the 5' end of the sense strand, or at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand; (C) the sense strand contains 2'-fluoro nucleotides at positions 9, 10, and 11, counting from the 5' end of the sense strand; or (D) the sense strand contains 2'-fluoro nucleotides at positions 11, 12, and 13, counting from the 3' end of the sense strand; (iv) the antisense strand comprises at least one, e.g., 2, 3, 4, 5, 6, 7 or more, DNA nucleotides, and optionally (A) the antisense strand comprises DNA nucleotides at positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand; (B) the antisense strand comprises DNA nucleotides at positions 2, 5, 7, 12, and 14, counting from the 5' end of the antisense strand; (C) the antisense strand comprises DNA nucleotides at positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand; or (D) the antisense strand comprises DNA nucleotides at positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand, and a 2'-fluoro nucleotide at position 14 of the antisense strand; (v) contains at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 2'-OMe nucleotide; (vi) the antisense strand comprises at least one 2'-OMe nucleotide, and optionally, the remaining nucleotides in the antisense strand are all 2'-OMe nucleotides; (vii) the sense strand comprises at least one 2'-OMe nucleotide, and optionally, the remaining nucleotides in the sense strand are all 2'-OMe nucleotides; (viii) contains at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, locked nucleic acid (LNA) or bridged nucleic acid (BNA) nucleotides; (ix) the antisense strand contains at least one, e.g., two, three, four, five, or more, LNA or BNA nucleotide; (x) the sense strand contains at least one, e.g., two, three, four, five, or more, LNA or BNA nucleotide; (xi) the dsRNA comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, cyclohexene nucleic acid (CeNA) nucleotide or analog thereof, and optionally, the CeNA nucleotide or analog thereof is and / or the strand of the dsRNA is prepared using a monomer selected from the group consisting of where: B is an optionally modified nucleobase; R is F, Cl, Br, I, H, protected OH, OMe, F, O-MOE, O-alkyl, O-alkene, O-alkyne, OC 16 , a branched lipid, or a protected aminoalkyl; R 1 are F, Cl, Br, I, H, protected OH, OMe, F, O-MOE, O-alkyl, O-alkene, O-alkyne, OC 16 , a branched lipid, a protected aminoalkyl, R' is H or CH 3 and PG is a protecting group. (xii) the antisense strand contains at least one, e.g., two, three, four, five, or more, CeNA nucleotides; (xiii) the sense strand contains at least one, e.g., two, three, four, five, or more, CeNA nucleotides; (xiv) comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, thermostabilizing modification; (xv) the antisense strand contains at least one, e.g., two, three, four, five, or more, thermostabilizing modification; (xvi) the sense strand contains at least one, e.g., two, three, four, five, or more, thermostabilizing modification; (xvii) contains at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, abasic nucleotides; (xviii) The antisense strand contains at least one, e.g., two, three, four, five, or more, abasic nucleotides; (xix) The sense strand contains at least one, e.g., two, three, four, five, or more, abasic nucleotides; (xx) containing at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 2'-deoxynucleotide; (xxi) The antisense strand contains at least one, e.g., two, three, four, five, or more, 2'-deoxynucleotide; (xxii) the sense strand contains at least one, e.g., two, three, four, five, or more, 2'-deoxynucleotide; (xxiii) containing at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, acyclic (e.g., unlocked nucleic acid (UNA) or glycol nucleic acid (GNA)) nucleotide; (xxiv) The antisense strand contains at least one, for example, 2, 3, 4, 5 or more, acyclic (e.g., unlocked nucleic acid (UNA) or glycol nucleic acid (GNA)) nucleotide; (xxv) The sense strand contains at least one, e.g., 2, 3, 4, 5, or more, acyclic (e.g., unlocked nucleic acid (UNA) or glycol nucleic acid (GNA)) nucleotide; (xxvi) containing at least one heat-destabilizing modification (e.g., an abasic nucleotide, a 2'-deoxynucleotide, an acyclic nucleotide (e.g., an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA) or an (S)-glycol nucleic acid (S-GNA)), a 2'-5' linked nucleotide (3'-RNA), a threose nucleotide (TNA), a 2' gem Me / F nucleotide, or a mismatch with the opposite nucleotide in the other strand); (xxvii) the antisense strand contains at least one thermodestabilizing modification; (xxviii) the antisense strand comprises at least one thermodestabilizing modification in the seed region of the antisense strand (i.e., positions 2-9 from the 5' end), and / or (xxix) The antisense strand contains a thermodestabilizing modification at least at one of positions 6, 7, or 8, counting from the 5' end of the strand, and optionally, the antisense strand contains a thermodestabilizing modification at position 7, counting from the 5' end of the strand. The dsRNA of claim 1.

10. An oligonucleotide comprising one or both of (a) and (b): (a) A 5'-terminal nucleoside which is a 2'-geminal substituted nucleoside of formula (II): and (b) at least one 2'-geminal substituted nucleoside of formula (I): In formula (I) and formula (II), X is O, S, C (R X ) 2 , or N(R XN ) and Each R X are independently hydrogen, halogen, or optionally substituted C 1～4 Alkyl, C 1～4 Haloalkyl, optionally substituted C 2～4 Alkenyl, or optionally substituted C 2～4 alkynyl, or both R X Overall, =O, =S, =N (R N ), or =CH 2 Forming R XN is hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 1 ~C 30 Alkoxy, C 1～4 Haloalkyl, optionally substituted C 2～4 Alkenyl, optionally substituted C 2～4 Alkynyl, optionally substituted C 1～30 Alkyl-CO 2 H, or a nitrogen protecting group, B is an optionally modified nucleobase; R a' is halogen, hydrogen, -OR a2 , -SR a3 , optionally substituted C 1～30 Alkyl, C 1～30 Haloalkyl, optionally substituted C 2～30 Alkenyl, optionally substituted C 2～30 Alkynyl, or optionally substituted C 1～30 Alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O) m CH 2 CH 2 OR a4 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH) n CH 2 CH 2 -R a5 , NHC(O)R a4 , to a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, a solid support, a linker covalently attached to a solid support, or an internucleoside linkage to a subsequent nucleoside; R a2 is a hydrogen or hydroxyl protecting group, R a3 is a hydrogen or sulfur protecting group, R a4 independently for each occurrence, H, C 1 ~C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R a5 and R a5 independently for each occurrence, an amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino; m is 1 to 50; n is 1 to 50; R b may be substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, or optionally substituted C 2～30 is alkynyl, R c is the bond to the internucleoside linkage to the subsequent nucleoside, hydrogen, halogen, -OR c2 , -SR c3 , optionally substituted C 1～30 Alkyl, C 1～30 Haloalkyl, optionally substituted C 2～30 Alkenyl, optionally substituted C 2～30 Alkynyl, or optionally substituted C 1～30 Alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O) r CH 2 CH 2 OR c4 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH) s CH2CH2-R c5 , NHC(O)R c4 , a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, a solid support, or a linker covalently attached to a solid support, wherein optionally at least R c or R a' is the bond to the internucleoside linkage to the subsequent nucleoside, R c2 is a hydrogen or hydroxyl protecting group, R c3 is a hydrogen or sulfur protecting group, R c4 independently for each occurrence, H, C 1 ~C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R c5 and R c5 independently for each occurrence, an amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino; r is 1 to 50; s is 1 to 50, R 4 is hydrogen, optionally substituted C 1～6 Alkyl, optionally substituted C 2～6 Alkenyl, optionally substituted C 2～6 Alkynyl, or optionally substituted C 1～6 Is it an alkoxy? Alternatively, R 4 and R a is, as a whole, 4'-C(R a11 R a12 ) v -Y-2' or 4'-YC (R a11 R a12 ) v -2', Y is -O-, -CH 2 -, -CH(Me)-, -C(CH 3 ) 2 -, -S-, -N (R a13 )-, -C(O)-, -C(S)-, -S(O)-, -S(O) 2 -, -OC(O)-, -C(O)O-, -N(R a13 )C(O)-, or -C(O)N(R a13 ) - and R a11 and R a12 are independently H, optionally substituted C 1 ~C 6 Alkyl, optionally substituted C 2 ~C 6 Alkenyl or optionally substituted C 2 ~C 6 is alkynyl, R a13 is hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 1 ~C 30 Alkoxy, C 1～4 Haloalkyl, optionally substituted C 2～4 Alkenyl, optionally substituted C 2～4 Alkynyl, optionally substituted C 1～30 Alkyl-CO 2 H, or a nitrogen protecting group, v is 1, 2 or 3, Alternatively, R 4 and R c C, together with the atoms to which they are attached, may be substituted 3～8 Cycloalkyl, optionally substituted C 3～8 forming a cycloalkenyl or an optionally substituted 3- to 8-membered heterocyclyl, R d is -CH(R d1 )-R d2 or -C(R d1 ) = CHR d2 and R d1 is hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted -C 2～30 alkenyl, or optionally substituted -C 2～30 is alkynyl, R d2 is the bond to the internucleoside linkage to the preceding nucleoside, R e is optionally substituted -C 2～6 Alkenyl-R e1 , optionally substituted C 1～6 Alkyl-R e1 , or optionally substituted -C 2～6 Alkynyl-R e1 and R e1 は、-P(O)(OR e4 ) 2 、-OR e2 、-SR e3 、-P(S)(OR e4 ) 2 、-P(S)(SR e5 )(OR e4 )、-P(S)(SR e5 ) 2 、-OP(O)(OR e4 ) 2 、-OP(S)(OR e4 ) 2 、-OP(S)(SR e5 )(OR e4 )、-OP(S)(SR e5 ) 2 、-SP(O)(OR e4 ) 2 、-SP(S)(OR e4 ) 2 、-SP(S)(SR e5 )(OR e4 )、または-SP(S)(SR e5 ) 2 であり、 R e2 is a hydrogen or oxygen protecting group, R e3 is a hydrogen or sulfur protecting group, Each R e4 are independently hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, or optionally substituted C 2～30 alkynyl, or oxygen protecting group; And each R e5 are independently hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, or optionally substituted C 2～30 alkynyl, or sulfur protecting group; provided that the nucleoside of formula (I) has the structure nucleosides, but where: R b is hydrogen or substituted or unsubstituted C 1 ~C 4 is alkyl, R c -OR is the bond to the internucleotide linkage to the subsequent nucleoside Ix where: R Ix H,-P(O)(Om) 2 ,-P(O)(OM)-OP(O)(OM) 2 , -P(O)(Oalkyl) 2 , -P(O)(Oalkyl)-O-P(O)(Oalkyl) 2 , -PO 3 H 2 , -PO 3 H.M., -P.O. 3 M 2 , -PO 2 SH 2 , -PO 2 SHM, -PO 2 SM 2 , -PO 3 M, or -PO 2 SM, protecting group, ligand, ligand-bearing monomer, -F, -(C r C 6 ) alkyl, -(C 2 ~C 6 ) allyl, -(C(R 3 ) 2 ) n OR 3 , -(C(R 3 ) 2 ) n SR 3 , -(C(R 3 ) 2 ) n N (R 3 ) 2 , -(C(R 3 ) 2 ) n C(O)N(R 3 ) 2 , -(C(R 3 ) 2 ) n O (C r C 6 ) alkyl, -(C(R 3 ) 2 ) n S (C r C 6 ) alkyl, -(C(R 3 ) 2 ) n O(C(R 3 ) 2 ) n N ((Ci~C 6 ) alkyl) 2 , -(C(R 3 ) 2 ) n ON ((Ci~C 6 ) alkyl) 2 , -C(O)R 3 , -C(O)R 3 C(O)H, -C(O)R 3 C(O)OH, -C(O)R 3 C(O)R 3 , -C(O)R 3 C(O)NR 3 -PO 2 , -P (OR 3 ) 2 , -P(N(R 3 ) 2 ) 2 , -P (OR 3 )N(R 3 ) 2 , or a linker, R 4 is H, R d is the bond to the internucleotide linkage to the preceding nucleoside, M represents, independently at each occurrence, an alkali metal or transition metal having a total charge of +1; and n is an integer from 1 to 4, and (i) The nucleoside of formula (II) has the structure nucleosides, but where: R b is hydrogen or substituted or unsubstituted C 1 ~C 4 is alkyl, R c -OR is the bond to the internucleotide linkage to the subsequent nucleoside Ix where: R Ix H,-P(O)(Om) 2 ,-P(O)(OM)-OP(O)(OM) 2 , -P(O)(Oalkyl) 2 , -P(O)(Oalkyl)-O-P(O)(Oalkyl) 2 , -PO 3 H 2 , -PO 3 H.M., -P.O. 3 M 2 , -PO 2 SH 2 , -PO 2 SHM, -PO 2 SM 2 , -PO 3 M, or -PO 2 SM, protecting group, ligand, ligand-bearing monomer, -F, -(C r C 6 ) alkyl, -(C 2 ~C 6 ) allyl, -(C(R 3 ) 2 ) n OR 3 , -(C(R 3 ) 2 ) n SR 3 , -(C(R 3 ) 2 ) n N (R 3 ) 2 , -(C(R 3 ) 2 ) n C(O)N(R 3 ) 2 , -(C(R 3 ) 2 ) n O (C r C 6 ) alkyl, -(C(R 3 ) 2 ) n S (C r C 6 ) alkyl, -(C(R 3 ) 2 ) n O(C(R 3 ) 2 ) n N((Ci-C 6 ) alkyl) 2 , -(C(R 3 ) 2 ) n ON ((Ci-C 6 ) alkyl) 2 , -C(O)R 3 , -C(O)R 3 C(O)H, -C(O)R 3 C(O)OH, -C(O)R 3 C(O)R 3 , -C(O)R 3 C(O)NR 3 -PO 2 , -P (OR 3 ) 2 , -P(N(R 3 ) 2 ) 2 , -P (OR 3 )N(R 3 ) 2 , or a linker, R 4 is H, R e is -CH 2 OR IIx where: R IIx -H, -P(O)(Om) 2 ,-P(O)(OM)-OP(O)(OM) 2 , -P(O)(Oalkyl) 2 , -P(O)(Oalkyl)-O-P(O)(Oalkyl) 2 , -PO 3 H 2 , -PO 3 H.M., -P.O. 3 M 2 , -PO 2 SH 2 , -PO 2 SHM, -PO 2 SM 2 , -PO 3 M, or -PO 2 SM, protecting group, ligand, or ligand-bearing monomer; M represents, independently at each occurrence, an alkali metal or transition metal having a total charge of +1; and n is an integer from 1 to 4.

11. 11. The oligonucleotide of claim 10, wherein the 5' terminal nucleotide is a 2'-geminal substituted nucleotide of formula (II) or (II').

12. (A) (i) The 2'-geminal substituted nucleoside of formula (II) is a nucleoside of formula (IIA): or formula (IIB): or or (ii) the 2'-geminal substituted nucleoside of formula (II') is a nucleoside of formula (IIA'): Or formula (IIB'): and / or (B) (i) The 2'-geminal substituted nucleoside of formula (I) is a nucleoside of formula (IA): or formula (IB): or or (ii) the 2'-geminal substituted nucleoside of formula (I') is a 2'-geminal substituted nucleoside of formula (IA'): or formula (IB'): Follow The oligonucleotide of claim 10. (i) X is O; (ii) (A) R a' But hydrogen, halogen, -OR a2 , optionally substituted C 1 ~C 30 Alkyl, optionally substituted C 1 ~C 30 Alkoxy, -O(CH 2 CH 2 O) m CH 2 CH 2 OR a4 , or -NH(CH 2 CH 2 NH) n CH 2 CH 2 -R a5 Or (B) R a' But halogen, hydrogen, -OR a2 , or optionally substituted C 1 ~C 30 Is it an alkoxy? (C) R a' But halogen, -OR a2 , or optionally substituted C 1 ~C 30 Is it an alkoxy? (D) R a' is F, OH or optionally substituted C 1 ~C 30 is alkoxy, or (E) R a' But amino or C 1 ~C 6 optionally substituted with alkoxy, C 1 ~C 30 is an alkoxy, (iii) (A) R b may be substituted C 1～6 Alkyl, C 1～6 Haloalkyl, optionally substituted C 2～6 Alkenyl, or optionally substituted C 2～6 is alkynyl, or (B) R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl or propargyl, or (C) R b is methyl, vinyl, ethynyl, allyl or propargyl; (iv) R 4 is hydrogen, optionally substituted C 1～6 Alkyl, optionally substituted C 2～6 Alkenyl, optionally substituted C 2～6 Alkynyl, or optionally substituted C 1～6 alkoxy, and optionally R 4 is hydrogen, (v) R d -CH(R d1 )-X d -R d2 That is, (vi)X d is O, (vii) R d1 is hydrogen or optionally substituted C 1 ~C 6 alkyl, and optionally R d1 is hydrogen; (viii) R e Ga-C 2～6 Alkenyl-R e1 or C 1～6 Alkyl-R e1 and C 1～6 Alkyl and C 2～6 Alkenyl may be optionally substituted; (xix) R e -CH=CHR e1 That is, (xx) R e1 -P(O)(OR e4 ) 2 , -OR e2 , or -OP (O) (OR e4 ) 2 That is, (xxi)R e2 is hydrogen or optionally substituted C 1 ~C 6 is alkyl, (xxii) a 2'-geminal substituted nucleoside of formula (I) is at least one of positions 2, 3, 4, 5, 6, 7, 8, 9 or 10, e.g., at least one of positions 5, 6, 7 and / or 8, counting from the 5' end of the oligonucleotide, and optionally a 2'-geminal substituted nucleoside of formula (I) is at position 7, counting from the 5' end of the oligonucleotide; (xxiii) further comprising a ligand linked to the oligonucleotide; (xxiv) containing only 2'-geminal substituted nucleosides of formula (I), (I'), (II) and (II'), (xxv) further comprising at least one modified internucleoside linkage; (xxvi) further comprising at least one modified nucleobase; (xxvii) further comprising at least one nucleoside modified at the 2' position, wherein the nucleotide modified at the 2' position is not a 2'-geminal nucleoside; (xxviii) The at least one nucleoside modified at the 2' position is a 2'-F or 2'-OMe nucleoside. (xxix) 10 to 50 nucleotides in length; (xxx) linked to a solid support; The oligonucleotide of claim 10. (i) The 2'-geminal substituted nucleoside of formula (II) is a 2'-geminal substituted nucleoside of formula (IIA): and During the ceremony, X is O, R a' is halogen (e.g., F, Cl, or Br), hydroxyl, optionally substituted C 1～30 Alkoxy (e.g., -(CH2 2 ) n CH 3 where n is 1 to 21, e.g., 1, 16, or -(CH 2 ) m -NH 2 where m is 2 to 10, for example 3 or 6, or -(CH 2 ) p -OMe, where p is 1-21, e.g., 1 or 2), or the bond to the internucleoside linkage to a subsequent nucleoside; R b may be substituted C 1～6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl) or halogen (e.g., F, Cl, or Br); R c is the bond or hydroxyl to the internucleoside linkage to the subsequent nucleoside, provided that R a' and R c is the bond to the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R e is -CH=CHR e1 where R e1 -P(O)(OR e4 ) 2 That is, (ii) The 2'-geminal substituted nucleoside of formula (II) is a 2'-geminal substituted nucleoside of formula (IIB): and During the ceremony, X is O, R a' is halogen (e.g., F), hydroxyl, optionally substituted C 1～30 Alkoxy (e.g., -(CH2 2 ) n CH 3 where n is 1 to 21, e.g., 1, 16, or -(CH 2 ) m -NH 2 where m is 2 to 10, for example 3 or 6, or -(CH 2 ) p -OMe, where p is 1-21, e.g., 1 or 2), or the bond to the internucleoside linkage to a subsequent nucleoside; R b may be substituted C 1～6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl); R c is the bond or hydroxyl to the internucleoside linkage to the subsequent nucleoside, provided that R a' and R c is the bond to the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R e is -CH=CHR e1 where R e1 -P(O)(OR e4 ) 2 That is, (iii) The 2'-geminal substituted nucleoside of formula (I) is a 2'-geminal substituted nucleoside of formula (IA): and During the ceremony, X is O, R a' is halogen (e.g., F, Cl, or Br), hydroxyl, optionally substituted C 1～30 Alkoxy (e.g., -(CH2 2 ) n CH 3 where n is 1 to 21, e.g., 1, 16, or -(CH 2 ) m -NH 2 where m is 2 to 10, for example 3 or 6, or -(CH 2 ) p -OMe, where p is 1-21, e.g., 1 or 2), or the bond to the internucleoside linkage to a subsequent nucleoside; R b may be substituted C 1～6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl) or halogen (e.g., F, Cl, or Br); R c is the bond or hydroxyl to the internucleoside linkage to the subsequent nucleoside, provided that R a' and R c is the bond to the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R d is the bond to the internucleoside linkage to the preceding nucleoside, However, R a' and R c is the bond to the internucleoside linkage to the subsequent nucleoside, or (iv) The 2'-geminal substituted nucleoside of formula (I) is a 2'-geminal substituted nucleoside of formula (IB): and During the ceremony, X is O, R a' is halogen (e.g., F, Cl, or Br), hydroxyl, optionally substituted C 1～30 Alkoxy (e.g., -(CH2 2 ) n CH 3 where n is 1 to 21, e.g., 1, 16, or -(CH 2 ) m -NH 2 where m is 2 to 10, for example 3 or 6, or -(CH 2 ) p -OMe, where p is 1-21, e.g., 1 or 2), or the bond to the internucleoside linkage to a subsequent nucleoside; R b may be substituted C 1～6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl) or halogen (e.g., F, Cl, or Br); R c is the bond or hydroxyl to the internucleoside linkage to the subsequent nucleoside, provided that R a' and R c is the bond to the internucleoside linkage to the subsequent nucleoside, R 4 is hydrogen, and R d is the bond to the internucleoside linkage to the preceding nucleoside, However, R a' and R c only one of which is the bond to the internucleoside linkage to the subsequent nucleoside, The oligonucleotide of claim 10.

15. 11. A double-stranded nucleic acid comprising a first oligonucleotide strand and a second oligonucleotide strand substantially complementary to the first strand, wherein the first strand and / or the second strand is the oligonucleotide according to claim 10. (i) the first strand and the second strand are independently 15 to 25 nucleotides in length; (ii) the first strand and / or the second strand have an overhang of 1 to 5 nucleotides at their respective 5' or 3' ends; (iii) only one of the first strand or the second strand has a two-nucleotide single-stranded overhang at its 5' or 3' end; (iv) only one strand has a single-stranded overhand of two nucleotides at its 3' end; and / or (v) the second strand comprises a ligand linked to the second strand; (vi) the first strand is substantially complementary to a target nucleic acid, and the double-stranded nucleic acid is capable of inducing RNA interference; The double-stranded nucleic acid of claim 15.

17. (i) the double-stranded RNA of any one of claims 1 to 9, wherein the strand is complementary to a target gene; (ii) the double-stranded RNA of any one of claims 15 to 16, wherein the first strand (e.g., the antisense strand) is complementary to a target gene; or (iii) the oligonucleotide of any one of claims 10 to 14, which is complementary to a target gene; A pharmaceutical composition for reducing the expression of a target gene in a subject, comprising any one of the following:

18. Compounds of formula (III) or (III'): During the ceremony, X is O, S, C (R X ) 2 , or N(R XN ) and Each R X are independently hydrogen, halogen, or optionally substituted C 1～4 Alkyl, C 1～4 Haloalkyl, optionally substituted C 2～4 Alkenyl, or optionally substituted C 2～4 alkynyl, or both R X Overall, =O, =S, =N (R N ), or =CH 2 Forming R XN is hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 1 ~C 30 Alkoxy, C 1～4 Haloalkyl, optionally substituted C 2～4 Alkenyl, optionally substituted C 2～4 Alkynyl, optionally substituted C 1～30 Alkyl-CO 2 H, or a nitrogen protecting group, B is an optionally modified nucleobase; R a is halogen, hydrogen, -OR a2 , -SR a3 , optionally substituted C 1～30 Alkyl, C 1～30 Haloalkyl, optionally substituted C 2～30 Alkenyl, optionally substituted C 2～30 Alkynyl, or optionally substituted C 1～30 Alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O) m CH 2 CH 2 OR a4 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH) n CH 2 CH 2 -R a5 , NHC(O)R a4 a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, a solid support, a linker covalently attached to a solid support, or a reactive phosphorus group; R a2 is a hydrogen or hydroxyl protecting group, R a3 is a hydrogen or sulfur protecting group, R a4 independently for each occurrence, H, C 1 ~C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R a5 and R a5 independently for each occurrence, an amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino; m is 1 to 50; n is 1 to 50; R b may be substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, optionally substituted C 2～30 alkynyl, or halogen; R 3 is hydrogen, halogen, -OR c2 , -SR c3 , optionally substituted C 1～30 Alkyl, C 1～30 Haloalkyl, optionally substituted C 2～30 Alkenyl, optionally substituted C 2～30 Alkynyl, or optionally substituted C 1～30 Alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O) r CH 2 CH 2 OR c4 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH) s CH2CH2-R c5 , NHC(O)R c4 a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, a solid support, a linker covalently attached to a solid support, or a reactive phosphorus group; R c2 is a hydrogen or hydroxyl protecting group, R c3 is a hydrogen or sulfur protecting group, R c4 independently for each occurrence, H, C 1 ~C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R c5 and R c5 independently for each occurrence, an amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino; r is 1 to 50; s is 1 to 50, R 4 is hydrogen, optionally substituted C 1～6 Alkyl, optionally substituted C 2～6 Alkenyl, optionally substituted C 2～6 Alkynyl, or optionally substituted C 1～6 Is it an alkoxy? Alternatively, R 4 and R a is, as a whole, 4'-C(R a11 R a12 ) v -Y-2' or 4'-YC (R a11 R a12 ) v -2' is formed, Y is -O-, -CH 2 -, -CH(Me)-, -C(CH 3 ) 2 -, -S-, -N (R a13 )-, -C(O)-, -C(S)-, -S(O)-, -S(O) 2 -, -OC(O)-, -C(O)O-, -N(R a13 )C(O)-, or -C(O)N(R a13 ) - and R a11 and R a12 are independently H, optionally substituted C 1 ~C 6 Alkyl, optionally substituted C 2 ~C 6 Alkenyl or optionally substituted C 2 ~C 6 is alkynyl, R a13 is hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 1 ~C 30 Alkoxy, C 1～4 Haloalkyl, optionally substituted C 2～4 Alkenyl, optionally substituted C 2～4 Alkynyl, optionally substituted C 1～30 Alkyl-CO 2 H, or a nitrogen protecting group, v is 1, 2 or 3, Alternatively, R 4 and R c C, together with the atoms to which they are attached, may be substituted 3～8 Cycloalkyl, optionally substituted C 3～8 forming a cycloalkenyl or an optionally substituted 3- to 8-membered heterocyclyl, R 5 is optionally substituted -C 2～6 Alkenyl-R 5a , optionally substituted C 1～6 Alkyl-R 5a , or optionally substituted -C 2～6 Alkynyl-R 5a and R 5a is a phosphorus group, -OR 5b , -SR 5c , hydrogen, a protected phosphorous group, a solid support, or a linker to a solid support, provided that R 3a , R 3 and R 5 only one of which is a linkage to a solid support, R 5b is H or a hydroxyl protecting group, and R 5c is H or a sulfur protecting group, However, R a , R 3 , and R 5a only one of is a solid support or a linkage to a solid support, However, R a , R 3 and R 5a Only one of the is reactive phosphorus(III), and However, the compound has the structure rather than a compound of where: R b is hydrogen or substituted or unsubstituted C 1 ~C 4 is alkyl, R c -OR Ix where: R Ix H,-P(O)(Om) 2 ,-P(O)(OM)-OP(O)(OM) 2 , -P(O)(Oalkyl) 2 , -P(O)(Oalkyl)-O-P(O)(Oalkyl) 2 , -PO 3 H 2 , -PO 3 H.M., -P.O. 3 M 2 , -PO 2 SH 2 , -PO 2 SHM, -PO 2 SM 2 , -PO 3 M, or -PO 2 SM, protecting group, ligand, ligand-bearing monomer, -F, -(C r C 6 ) alkyl, -(C 2 ~C 6 ) allyl, -(C(R 3 ) 2 ) n OR 3 , -(C(R 3 ) 2 ) n SR 3 , -(C(R 3 ) 2 ) n N (R 3 ) 2 , -(C(R 3 ) 2 ) n C(O)N(R 3 ) 2 , -(C(R 3 ) 2 ) n O (C r C 6 ) alkyl, -(C(R 3 ) 2 ) n S (C r C 6 ) alkyl, -(C(R 3 ) 2 ) n O(C(R 3 ) 2 ) n N ((Ci~C 6 ) alkyl) 2 , -(C(R 3 ) 2 ) n ON ((Ci~C 6 ) alkyl) 2 , -C(O)R 3 , -C(O)R 3 C(O)H, -C(O)R 3 C(O)OH, -C(O)R 3 C(O)R 3 , -C(O)R 3 C(O)NR 3 -PO 2 , -P (OR 3 ) 2 , -P(N(R 3 ) 2 ) 2 , -P (OR 3 )N(R 3 ) 2 , or a linker, R 4 is H, R 5x H,-P(O)(Om) 2 ,-P(O)(OM)-OP(O)(OM) 2 , -P(O)(Oalkyl) 2 , -P(O)(Oalkyl)-O-P(O)(Oalkyl) 2 , -PO 3 H 2 , -PO 3 H.M., -P.O. 3 M 2 , -PO 2 SH 2 , -PO 2 SHM, -PO 2 SM 2 , -PO 3 M, or -PO 2 SM, protecting group, ligand, or ligand-bearing monomer; M represents, independently at each occurrence, an alkali metal or transition metal having a total charge of +1; and n is an integer from 1 to 4.

19. a. The compound of formula (III) is a compound of formula (IIIA): or b. The compound of formula (III') is a compound of formula (IIIA'): That is, 19. The compound of claim 18.

20. a. The compound of formula (III) is a compound of formula (IIIB): or b. The compound of formula (III') is a compound of formula (IIIB'): That is, 19. The compound of claim 18.

21. 20. The compound of claim 18, wherein the reactive phosphorus(III) group is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester.

22. The reactive phosphorus(III) group is -OP(OR P ) (NR P2 ) 2 , -OP(SR P ) (NR P2 ) 2 , -OP(O)(OR P ) (NR P2 ) 2 , -OP(S)(OR P ) (NR P2 ) 2 , -OP(O)(SR P ) (NR P2 ) 2 , -OP(O)(OR P ) H, -OP(S)(OR P ) H, -OP(O)(SR P ) H, -OP (O) (OR P ) R P3 , -OP(S)(OR P ) R P3 , or -OP(O)(SR P ) R P3 where: R P may be substituted C 1～6 is alkyl, and Each R P2 are independently optionally substituted C 1～6 Is it alkyl? Or, both R P2 together with the nitrogen atom to which they are attached form an optionally substituted 3- to 8-membered heterocyclyl; or Or, R P and R P2 together with the atoms to which they are attached form an optionally substituted 4- to 8-membered heterocyclyl; and R P3 may be substituted C 1 ~C 30 Alkyl, optionally substituted C 2 ~C 30 Alkenyl, or optionally substituted C 2 ~C 30 is alkynyl, Optionally, The reactive phosphorus(III) group is -OP (OR P ) (NR P2 ) 2 That is, 19. The compound of claim 18.

23. (i) R P is substituted with cyano or -SC(O)Ph, 1～6 alkyl, and optionally R P Ga-CH 2 CH 2 CN, (ii) each R; P2 is independently methyl, ethyl, propyl, or isopropyl, and optionally each R P2 is isopropyl; (iii) R P3 may be substituted C 1 ~C 6 is alkyl, 23. The compound of claim 22.

24. (i) X is O. (ii) (A) R a But halogen, hydrogen, -OR a2 , or optionally substituted C 1 ~C 30 Is it an alkoxy? (B) R a But halogen, -OR a2 , or optionally substituted C 1 ~C 30 Is it an alkoxy? (C) R a is F, Cl, OH or optionally substituted C 1 ~C 30 is alkoxy, or (D) R a But amino or C 1 ~C 6 optionally substituted with alkoxy, C 1 ~C 30 is an alkoxy, (iii) (A)R b may be substituted C 1～6 Alkyl, C 1～6 Haloalkyl, optionally substituted C 2～6 Alkenyl, optionally substituted C 2～6 alkynyl, or halogen; (B) R b is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl or propargyl, or (C) R b is methyl, vinyl, ethynyl, allyl or propargyl; (iv) R 3 H, halogen, OR c2 , a reactive phosphorus group, or a linker to a solid support, preferably R 3 is a reactive phosphorus group or a linkage to a solid support, more preferably R 3 is a reactive phosphorus(III) group; (v) R 4 is hydrogen, optionally substituted C 1～6 Alkyl, optionally substituted C 2～6 Alkenyl, optionally substituted C 2～6 Alkynyl, or optionally substituted C 1～6 alkoxy, and optionally R 4 is hydrogen, (vi)R 5 may be substituted -C 2～6 Alkenyl-R 5a or C 1～6 Alkyl-R 5a and preferably R 5 -CH=CHR 5a and optionally, (A) R 5a is a phosphorus group or -OR 5b Or (B) R 5a is a phosphorus group, or (C) R 5a が、-P(O)(OR e4 ) 2 、-P(S)(OR e4 ) 2 、-P(S)(SR e5 )(OR e4 )、-P(S)(SR e5 ) 2 、-OP(O)(OR e4 ) 2 、-OP(S)(OR e4 ) 2 、-OP(S)(SR e5 )(OR e4 )、-OP(S)(SR e5 ) 2 、-SP(O)(OR e4 ) 2 、-SP(S)(OR e4 ) 2 、-SP(S)(SR e5 )(OR e4 )、または-SP(S)(SR e5 ) 2 であり、 R e2 is a hydrogen or oxygen protecting group; R e3 is a hydrogen or sulfur protecting group; Each R e4 are independently hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, or optionally substituted C 2～30 alkynyl, or an oxygen protecting group, and Each R e5 are independently hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, or optionally substituted C 2～30 alkynyl, or sulfur protecting group, 20. The compound of claim 19.

25. (i) A compound of formula (IIIa): and During the ceremony, X is O, R a is halogen (e.g., F), hydroxyl, protected hydroxyl, optionally substituted C 1～30 Alkoxy (e.g., -(CH2 2 ) n CH 3 where n is 1 to 21, e.g., 1, 16, or -(CH 2 ) m -NH 2 where m is 2 to 10, for example 3 or 6, or -(CH 2 ) p -OMe, where p is 1-21, e.g., 1 or 2), a reactive phosphorus(III) group, a solid support, or a linker covalently attached to a solid support; R b may be substituted C 1～6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl); R 4 is hydrogen, R 3 is a reactive phosphorus(III) group, a solid support, a linker covalently attached to a solid support, a hydroxyl, or a protected hydroxyl, provided that R a and R 3 is a reactive phosphorus(III) group, a solid support, or a linker covalently attached to a solid support, and R 5 is -CH=CHR 5a where R 5a -P(O)(OR 5e ) 2 and each R 5e are independently hydrogen or optionally substituted C 1～30 is alkyl, (ii) The compound of formula (III) is a compound of formula (IIIB): and During the ceremony, X is O, R a is halogen (e.g., F, Cl, Br), hydroxyl, protected hydroxyl, optionally substituted C 1～30 Alkoxy (e.g., -(CH2 2 ) n CH 3 where n is 1 to 21, e.g., 1, 16, or -(CH 2 ) m -NH 2 where m is 2 to 10, for example 3 or 6, or -(CH 2 ) p -OMe, where p is 1-21, e.g., 1 or 2), a reactive phosphorus(III) group, a solid support, or a linker covalently attached to a solid support; R b may be substituted C 1～6 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, vinyl, ethynyl, allyl, or propargyl, preferably methyl) or halogen (e.g., F, Cl, Br); R 4 is hydrogen, R 3 is a reactive phosphorus(III) group, a solid support, a linker covalently attached to a solid support, a hydroxyl, or a protected hydroxyl, provided that R a and R 3 is a reactive phosphorus(III) group, a solid support, or a linker covalently attached to a solid support, and R 5 is -CH=CHR 5a where R 5a -P(O)(OR 5e ) 2 and each R 5e are independently hydrogen or optionally substituted C 1～30 is alkyl, 19. The compound of claim 18.

26. R 3 is a reactive phosphorus group, and optionally R 3 But -OP (OR P )N(R P2 ) 2 , -OP(SR P )N(R P2 ) 2 , -OP(O)(OR P )N(R P2 ) 2 , -OP(S)(OR P )N(R P2 ) 2 , -OP(O)(SR P )N(R P2 ) 2 , -OP(O)(OR P ) H, -OP(S)(OR P ) H, -OP(O)(SR P ) H, -OP (O) (OR P ) R P3 , -OP(S)(OR P ) R P3 , or -OP(O)(SR P ) R P3 26. The compound of claim 25, wherein:

27. R 3 -OP (OCH 2 CH 2 CN)N(iPr) 2 26. The compound of claim 25, wherein:

28. R 3 is a hydroxyl or a protected hydroxyl, or R 3 is a solid support or a linker covalently attached to a solid support; 26. The compound of claim 25.

29. R a is F, hydroxyl, protected hydroxyl, or optionally substituted C 1～30 Alkoxy (e.g., -(CH2 2 ) n CH 3 where n is 1 to 21, e.g., 1, 16, or -(CH 2 ) m -NH 2 where m is 2 to 10, for example 3 or 6, or -(CH 2 ) p 26. The compound of claim 25, wherein p is -OMe, where p is 1 to 21, for example 1 or 2.

30. selected from the group consisting of: wherein m is 2 to 10 (e.g., 3, 6) and n is 1 to 21 (e.g., 1, 16); 19. The compound of claim 18.

31. The compound of claim 18, selected from the group consisting of:

32. The compound of claim 18, selected from the group consisting of:

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